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The primary structure of guinea pig β2-microglobulin
Mokcular @ergamon
IrnrnU~~l~Y, Press Ltd.
Vol. I?, pp. 1493-1505. 1980. Printed in Great
0161-SS#j8O~~ZOl-i493
102.00/O
Britain
THE PRIMARY STRUCTURE OF GUINEA PIG P2-MICROGLOBULIN PAUL B. WOLFE and JOHN J. CEBRA Department of Biology, The Johns Hopkins University, Baltimore, MD 21218, U.S.A. (Received 3 April 1980)
Abstract-The complete primary structure of guinea pig b2-microglobulin has been determined. It is 99 residues long and shares approximately 70% of its sequence with human and rabbit p2m. Heterogeneity at the carboxyl-terminus gives rise to two forms of the molecule: one bearing a COOH-terminal lysyl residue and one which lacks this residue. Evidence suggests that the two forms arise due to proteolysis, but multiple forms offl2m are not ruled out. A model of the three-dimensional structure is presented and used to evaluate amino acid substitutions among microglobulins. A high degree of conservation is seen among residues responsible for maintenance of fl-structure. This is especially true for residues occurring in a presumptive four-chain layer and suggests that this is the face which interacts with histocompatibility antigens.
INTRODUCTION
Interest in 82-microglobulin (/?2m) has stemmed from two general observations. It has clear structural homology to the constant regions of immunoglobulins (Smithies & Poulik, 1972a; Peterson et al., 1972). This similarity has prompted consideration of a close evolutionary relationship between these molecules (Smithies & Poulik, 1972a). The obse~ation of more general interest has been that B2m is a subunit of both major and minor histocompatibility antigens in the cell membrane. Human (HLA), mouse (H-2K and H-2D) and guinea pig (GPLA) major histocompatibility antigens (Grey et al., 1973; Peterson et al., 1974; Silver & Hood, 1974; Rask et al., 1974; Schwartz et al., 1976), as well as the minor histocompatibility antigens of the mouse (Tla, Qa-2 and H-Y) and their analogs in the guinea pig (Ostberg et al., 1975; Vitetta et al., 1975; Vitetta et al., 1976; Fellous et al., 1978; Michaelson et al., 1977; Schwartz et al., 1978) are paired with p2m. Thus, ,!32m may be a common subunit of all major transplantation antigens and structurally related cell surface molecules. The precise roles played by these highly polymorphic glycoproteins are unknown. Their appearance as differentiation antigens and virus receptors (Helenius et al., 1978) and their participation in T cell-mediated immunity (Doherty et al., 1976) suggest that they perform a variety of biological functions. In contrast, /32m remains the essentially invariant component, not only within this class of molecules, but across species lines as well.
Consequently, study of the structure and function of fi2rn may provide insight into the roles of its more complex partner. We herein describe the isolation of guinea pig ~2-mi~roglobulin and the determination of its primary structure. Microglobulins from a variety of species, including guinea pig, have been described (Smithies & Poulik, 1972b; Poulik et al., 1975; Becker et al., 1977; CigCn et al., 1978; Winkler & Sanders, 1977). The complete primary structures of human and rabbit p2m are published (Cunningham et al., 1973; Gates et al., 1979) and several partial sequences of this molecule from other species have appeared as well (Smithies & Poulik, 19726; Poulik e.! al., 1975; Becker et al., 1977; CigCn et al., 1978). These data establish the sequence homology of p2m with immunoglobulin constant region domains and demonstrate a high degree of structural similarity among microglobulins. Our results extend those observations by providing a third complete sequence for comparison. A preliminary report and partial sequence have appeared (Wolfe & Cebra, 1978; Cebra et al., 1977).
MATERIALS AND METHODS
Purification of guinea pig /?2m was based partly on procedures described for purification of human and rabbit p2m (Berggard & Bearn, 1968; Berggard, 1974). Six to twelve strain 2, strain 13 or Hartley guinea pigs, raised in our own colony, were given subcutaneous injections
1493
1494
PAUL
B. WOLFE
of potassium chromate (10 mg/kg) in saline. The animals were kept in metabolism cages (3-6 animals per cage) and urine was collected for seven days following administration of the poison. No deaths resulted directly from this treatment and animals were frequently used more than once after a suitable recovery period (3-4 weeks). Each 24-hr specimen was frozen immediately after collection into a container immersed in a dry ice/acetone bath. On average, six animals yielded one liter of urine over seven days. After collection, the urine was thawed and
and JOHN
J. CEBRA
centrifuged to remove insoluble material. The resulting supernatant was dialysed in the cold against several changes of phosphate-buffered saline at pH 7.3. Urinary proteins were concentrated and a crude fi2m-containing precipitate was obtained by the addition of cold, saturated ammonium sulfate to 60’/,, saturation. The suspension was stirred in the cold overnight, then allowed to settle so that the majority of the supernatant could be drawn off by siphon. The remainder was centrifuged to collect the precipitate, which was dissolved in a minimum volume, dialysed against IO mM Tris-HCl,
b
a 2.4 -
:
2.4 E = 20
20-
0 a,
0 :
N
16-
I6
w
:
ii
1.2-
a m
Z g
1.2
: m 0.8
0.8-
:
:
a 0.4-
I1
40
60
80
FRACTION
100 NUMBER
120
140
I6(
I
20
I
I
40
FRACTION
2’
III
1
60
I
80
I
I
I
100
I
120
NUMBER
d
FRACTION
NUMBER
Fig. 1. Purification of guinea pig /?2-microglobulin. (a) Concentrated urinary proteins fractionated on Sephadex G-100; (b) fraction 4 from G-100 on Sephadex G-50 in urea and formic acid; (c) fraction 2 from G-SO on Sephadex G-75 in urea and formic acid; (d) SDS-polyacrylamide gel electrophoresis: column 2, whole urinary proteins; column 3, G-100 fraction 4; column 4, G-50 fraction 2; column 5, G-75 fraction 2 columns 1 and 6, molecular weight standards. Lactoperoxidase = 77,500 daltons; ovalbumin = 43,500 daltons; trypsin = 23,800 daltons; and lysozyme = 12,000 daltons.
Guinea Pig j2-Microglobulin
pH 8.0, containing 1 M NaCl and 0.05% NaN,, and chromatographed on a column of Sephadex G-100 (3.2 x 180 cm) equilibrated in the same buffer. It was found that fraction 4 (Fig. la) contained material of the appropriate molecular weight as assessed by SDS-polyacrylamide gel electrophoresis (Fig. Id), by the elution volume of a radiolabeled sample of rabbit P2m (data not shown) and by comparison to published observations using this chromatography system (Berggard, 1974). The material in this fraction was pooled, desalted on a column of Sephadex G-50, coarse (3 x 48 cm), equilibrated in 50 mM formic acid, and lyophilized. The lyophilized material was dissolved in 8 M urea which was 0.1 M in formic acid and chromatographed on a column of Sephadex G-50, superfine (2.5 x 180 cm), equilibrated in the same solvent. Fraction 2 (Fig. lb) contained material of the appropriate molecular weight as judged by SDS-polyacrylamide gel electrophoresis. This pool was desalted and lyophilized as before, subjected to total reduction and alkylation with iodoacetic acid (Birshtein et al., 1971) and chromatographed on a column of Sephadex G-75 (2.5 x 180 cm) in 8 Murea, 0.1 Min formic acid. Fraction 2 (Fig. lc) contained material of 11,500 daltons without significant contamination (Fig. Id). Amino acid analysis and N-terminal sequence analysis both indicated the presence of a homogeneous product. Automated sequential degradation
Carboxymethylated fi2m and peptides derived from it were submitted to automatic sequential degradation in a Beckman 890-B sequencer. The peptide program (111374, Beckman Instruments Corp.), using dimethylallylamine as a solvent buffer, was employed for all sequence determinations. For peptides which were small and/or hydrophobic, 50 ,ug of polybrene (Aldrich Chemicals, Milwaukee, WI) was used as a carrier. Polybrene was subjected to two full cycles of Edman degradation prior to the addition of peptide. Identification of the amino acid residues at each position was accomplished by procedures previously described (Birshtein et al., 1971). PTH derivatives of amino acids were also identified by thin layer chromatography on polyamide sheets (Summers et al., 1973) and by calorimetric methods (Yamada & Itano, 1966; Easley, 1965). Enzymic digestion of p2m
Carboxymethylated
/?2m (l-l .5 pmol) was
1495
digested at 37°C in 0.5% ammonium bicarbonate by the addition of trypsin (Code: TRTPCK, Worthington Biochemical Corp; Freehold, NJ) at an enzyme to substrate ratio of 1:200 (w/w) at hourly intervals for 4 hr. The reaction was allowed to continue for an additional 4 hr at 37°C without further addition of the enzyme. Carboxymethylated /32m (0.5-l pmol) was digested with a-chymotrypsin (Worthington Biochemical Corp.) under conditions identical with those for trypsin digestion. Carboxypeptidase A and carboxypeptidase B were used as previously described (Turner & Cebra, 1971). Carboxypeptidase Y (Worthington Biochemical Corp.) was used according to Hayashi et al. (1973). Succinylation
Carboxymethylated /?2m (0.5 pmol) was succinylated using a modification of the method suggested by Klotz (1967). The protein was dissolved in 5 ml of 6 M guanidine, 0.1 M borate, pH 9.1. Solid succinic anhydride, lOO-fold molar excess over available amino groups, was added slowly with stirring at room temperature. The pH was maintained by dropwise addition of 1 N NaOH. The reaction was continued for 30 min after the last addition of anhydride. Excess reagents were removed by dialysis against 0.1 M formic acid and the product was lyophilized. The extent of derivatization was checked by digesting a small sample (2-5 nmol) with trypsin (4 hr at 37°C) followed by digestion with carboxypeptidase B (4 hr at 37°C). The amount of lysine released as determined by amino acid analysis was always less than 1% of that obtained after acid hydrolysis of an equal amount of underivatized /?2m. Cyanogen bromide cleavage
Succinylated b2m (0.3 ,umol) was dissolved in 2 ml of 70% formic acid and chilled to 4°C. Quantitative cleavage at the single methionyl residue was achieved by addition of solid cyanogen bromide, then allowing the mixture to stand at 4°C for 24 hr (Birshtein et al., 1971). The reaction was terminated by addition of 10 vol. water followed by lyophilization. Other methods
Preparative high voltage paper electrophoresis was performed according to Turner & Cebra (1971). SDS-polyacrylamide gel electrophoresis was carried out as described by Laemmli (1970).
1496
PAUL 10
B. WOLFE
and JOHN
J. CEBRA 40
30
20
“LHAPR”PY~SRHPAENGK~~~*~~~“~~~~~~~~~~~~~~~~~~~~~“~ .____________________~~~~~~~~~~~~ T2 TI T3 T4 . --_~-_---r-_* ____________________----------
50
T5
--_
+GP T7 ;e T6-T7
4_-d-d Chl
.
c
Ch4
Ch3
-.
*
A3~~,,~~~~~~~~~~~~~~~~~~~~
Ch 2
A3
-
T6-T7-T6o
T8b
TS
T7
_
__
_
__
_
TII
TIO
___________
-
L-m Tllo
-D
T6-T7
_ v-_-4
Ch6
Ch7
Ch6 -e-d___
__-a__-
ChS
a__
Ch IO ________
Ch12
CNBr
.
_____4________
_---___
l
A3
_-.____--_-_______
A4
Fig. 2. A schematic representation of the procedure and resulting data used to deduce the sequence of guinea pig P2-microglobulin. Symbols used are: (+), removal of a residue by automatic Edman degradation; (-), identification of a residue by digestion with carboxypeptidase A; (-), identification of a residue by digestion with carboxypeptidase B.
Staining
procedures
were those of Fairbanks
et
al. (1971). RESULTS
Amino
terminal sequence of p2m
Automated sequential degradation of carboxymethylated guinea pig p2m (0.5 pmol) permitted identification of amino acids at 32 consecutive positions (Fig. 2). No sequence heterogeneity was observed among the first 25 residues in microglobulins obtained from Hartley, strain 2 or strain 13 animals. Carboxypeptidase
digestion of P2m
Digestion of carboxymethylated p2m with carboxypeptidases A, B or Y under nondenaturing conditions failed to release amino acids above control levels observed in the absence of substrate. Digestion with carboxypeptidase Y (CPY) in the presence of 6 M urea also failed to release carboxyl-terminal amino acids. However, a contaminating endopeptidase
activity in the CPY quantitatively split p2m between residues 65 and 66, which allowed the CPY to act efficiently at this site. Digestion with carboxypeptidase B (CPB) in the presence of 0.1% sodium dodecyl sulfate released from 0.5 to 0.8 moles of lysine per mole of substrate, depending on the preparation of 82m. Tryptic peptides Digestion of carboxymethylated b2m with trypsin and subsequent fractionation of tryptic peptides resulted in the isolation of a set of peptides which accounted for its entire amino acid sequence. Peptides Tl-T5, T7, and T9-Tl l were isolated as conventional tryptic fragments. Two partial digest products and one unexpected cleavage site resulted in the isolation of several related tryptic fragments. Peptide T6 is free lysine, which was not isolated. However, a variant of T7 (named T6-T7) was recovered with a lysyl residue at its amino-terminus. Peptide T8 was never isolated intact. Instead, peptides T8a and T8b, resulting from a chymotryptic-like
Guinea Pig /32-Microglobulin T6b T6-T7-T6a I
T4 I
T3 t
T9 t
T2 t
1497
TI t
TIO
1 56
2.8
2.4
d
0
0.4
60
00
100
120
FRACTION
140
160
160
200
NUMBER
Fig. 3. Gel filtration of tryptic peptides of fl2-microglobulin on a column of Sephadex G-50, superfine (2.5 x lSOcm), equilibrated in 50 mMammonium hydroxide. The solid line indicates absorbance at 215 nm. The dashed line indicates radioactivity originating from [3H]-carboxymethyl-cysteinyl residues. Fraction volume is 5.0 ml.
T7 ‘1
T4+Tllo
I
40
T6-T7
I
60 FRACTION
81,
I
80
100
T9 T3 T5
,
I
120
I
(
140
NUMBER
Fig. 4. Ion-exchange chromatography of tryptic peptides of /I2-microglobulin soluble at pH 3.1 on a column of sulfonated polystyrene (Beckman PA-35). The column was operated at 56°C with a linear 300 ml gradient of pyridinium acetate buffers, W-200 mM in pyridine, pH 3.1-5.4, which were 5% in I-propanol. The solid line indicates the relative fluorescence monitored at 480 nm following reaction with fluorescamine.
PAUL
1498
B. WOLFE
and JOHN
cleavage, were always observed. Peptide T8a was recovered as T6-T7-T8a. Apparently trypsin did not quantitatively cleave the lysyl-isoleucyl bond joining T6 and T7 or the lysyl-aspartyl bond linking T7 and T8a. Finally, a variant of the carboxyl-terminal peptide, Tl 1, was isolated. Peptide Tl la differs from Tl 1 only in that it lacks a C-terminal lysyl residue. Degradation prior to the isolation of the intact molecule is a plausible explanation for the appearance of this peptide. The tryptic digest was initially fractionated on a column of Sephadex G-50 as illustrated in Fig. 3. Pool 1 yielded peptides T6-T7-T8a and T8b after additional fractionation by gel chromatography (data not shown). Pools 2 and 3 contained peptides T4 and T3, respectively, and did not require further treatment. The other peptides eluted from this column were purified by high voltage paper el~trophoresis. Peptide T9 was isolated by this method from pool 4, T2 from pool 5, Tl from pool 6 and TlO from pool 7. Tryptic peptides soluble at pH 3.1 were also fractionated by ion-exchange chromatography. Figure 4 shows the results of fractionation on a sulfonated polystyrene resin (PA-35, Beckman Instruments Corp.). Peptides T3, T5, T6-T7, T7 and Tll were isolated directly by this method from pools 10, 11, 7,2 and 9, respectively. Pool 4 contained an equimolar mix of T4 and Tl 1a. Gel filtration of this mixture on Sephadex G-25, superfine (2 x 35 cm), in 60 mA4 HCl resolved these two peptides. Sequences and partial sequences of some of the tryptic peptides were determined as shown in
Table Tl LYS His Arg CMCys Asp Thr Ser Glu Pro GUY Ala Val Met Ile Leu Tyr Phe Trp
0.7 0.8
T2
1. Amino
Fig. 2. The amino acid compositions of all of the tryptic peptides are shown in Table 1. Chymotryptic
of the tryptic
peptides T6-T7-T8a
T4
T5
T&T7
T7
1.1 0.9
1.0 0.9
1.2
1.9
1.1
1.1
A:f
0.9 1.0 1.3
2.0
1.0 0.9
1.1 0.9 1.0 0.9
n
1.0 3.9 2.0 1.0 2.1 1.9 2.1 1.0 1.8
2.1 2.3 1.3
2.9 2.3 2.2
from ~2-microglobulin TSb
1.7
1.1
1.0
peptides
Peptides from a ~hymotryptic digest of carboxymethylated /?2m were fractionated by ion-exchange chromatography on a column of Beckman PA-35 as shown in Fig. 5. Pool 1 contained an assortment of fragments which eventually yielded peptide Ch9 after additional resolution by gel filtration (data not shown). Peptides Ch3 and Ch4 were resolved after chromatography of pool 2 on a column of Sephadex G-25, superfine (2 x 35 cm), equilibrated in 60 mA4 HCl. Pools 3 and 4 contained peptides Ch12 and Ch7, respectively, and required no further purification. Pool 7 yielded peptides Ch6 and ChlO after fractionation on a column of Sephadex G-25 (2.5 x 180 cm) in 50 mM ammonium bicarbonate which had been adjusted to pH 10 with ammonium hydroxide. Peptides in pools 6 and 8 were purified by preparative high voltage paper electrophoresis. Peptide Ch8 was isolated from pool 6, peptide Chl from pool 8. The amino acid compositions of the chymotryptic peptides are shown in Table 2. of sequence analysis of The results chymotryptic peptides are summarized in Fig. 2. Peptide Chl verifies an overlap between tryptic peptides Tl and T2, peptide Ch6 confirms the overlap between T7 and T8a, and peptide ChlO establishes the overlap between T8b and T9. Peptides Ch8 and Ch9 together confirm the sequence of T8b.
T3
1
1.0 1.1
acid compositions
J. CEBRA
4.2 1.0 2.6 1.5
0.9 1.0 1.3 3.7 1.1 2.1 1.4 1.2
T9
T10
1.1 0.7
0.9
0.9 1.7 1.1 1.3
Tlla
Tll 0.9
1.9
2.0
1.0
0.9
i-
+
0.9 1.1 0.8 0.9 1.2
1.3 0.6 1.2 1.4
0.9
1.3
1.2 0.5
2.0 1.2
I .o 1.2 I.0 I .8 +
2.3 1.1 1.2
1.0
1.1
0.9 1.0
1.0
Guinea Pig /72-Microglobulin ch9
,t
0112Ch7
Ch3+Ch4
t
t
Chg Ch6+ChlOCh I
t
t t
,,,,,
,
I
,
20
I
1499
56
1
Jl,
I,
I
40
60 FRACTION
00
100
6 *
120
140
NUMBER
Fig. 5. Ion-exchange chromatography of chymotryptic peptides of /?2-microglobulin on a column of sulfonated polystyrene (Beckman PA-35). The column was operated at 56°C with a linear 300 ml gradient of pyridinium acetate buffers, SO-200 mMin pyridine, pH 3.1-5.4, which were 5% in 1-propanol. The solid line indicates the relative fluorescence monitored at 480 nm following reaction with fluorescamine.
Sequence following cyanogen bromide cleavage
Carboxymethylated /32m was treated with succinic anhydride to block all primary amines, then cleaved with cyanogen bromide. Cleavage after the single methionyl residue generated a new amino terminus and the products of the reaction were directly subjected to automated sequence analysis without further treatment. Results of this analysis are shown in Fig. 2. The sequence
begins at Ser-52 and by necessity assigns the methionine to residue position 51. It also confirms the carboxyl-terminal sequence of tryptic peptide T7 and establishes an overlap across T7, T8a and T8b. Succinylated tryptic and chymotryptic peptides
Succinylated /?2m was digested with trypsin in order to obtain peptides resulting from cleavage
Table 2. Amino acid compositions of the chymotryptic peptides from /32-microglobulin Chl
Ch3
Ch4
Ch7
Ch8
Ch9
ChlO
1.2
LYS
His Arg CMCys Asp Thr Ser Glu Pro Gly Ala Val Met Ile Leu Tyr Phe Trp
Ch6
0.8 1.0
1.0
1.0 1.0 1.0
1.1 0.9
0.9 3.1 2.2 1.1
1.5
1.0 0.9 0.8
1.7 0.9 1.4 1.0
2.1
2.1 1.1
1.1
0.8 2.1
2.0
0.9 1.0
0.9
2.2
0.8 1.0 1.0 3.1 1.0 0.9 1.1 1.3
1.0 1.0 1.0
1.0 1.2
0.8 +
Ch12
1.0 1.0
1.0 1.1
1500
PAUL
B. WOLFE
400
and JOHN
J. CEBRA
600 EFFLUENT
VOLUME
(m/s)
Fig. 6. Gel filtration of tryptic peptides of succinylated P2-microglobulin on a column of Sephadex G-50, superfine (2.5 x 180 cm), equilibrated in 50 mM ammonium bicarbonate adjusted to pH 10.0 with ammonium hydroxide. The solid line indicates the absorbance at 280 nm. The dashed line indicates radioactivity originating from [3H]-carboxymethylcysteinyl residues. Fraction volume is 5.0 ml.
after arginyl residues. These peptides were fractionated on a column of Sephadex G-SO as shown in Fig. 6. Material in pool 1 was found to correspond in amino acid composition to the peptide extending from His-13 to Arg-81 and was designated peptide A3. Apparently trypsin failed to cleave the succinylated molecule between T8a and T8b as it did the underivatized protein. Pool 2 contained the peptide designated A4 which extends from Val-82 to the carboxylterminal end. Automated sequential degradation of A4 resulted in the determination of its complete sequence which established the order of tryptic peptides T9, TlO and Tl 1. As expected, pool 3 contained a mixture of the N-terminal hexapeptides Al and A2 (Tl and T2). DISCUSSION
Our initial approach to the purification of guinea pig P2-microglobulin was based on procedures described for the isolation of human and rabbit /I2rn (Berggard & Bearn, 1968; Berggard, 1974). These methods employ ionexchange or electrophoretic techniques in isolating this protein. We found methods involving separation by net charge to be impractical due to the presence of a contaminant which was yellow-brown in color and conferred charge heterogeneity to most of the urinary proteins. The contaminant remained associated with /?2m in the presence of urea and formic acid.
A suspected covalent association was confirmed when, after reduction and alkylation of cysteinyl residues, the material was easily removed by dialysis. No attempts were made to further characterize the contaminating material. With the exception of ammonium sulfate precipitation, used to concentrate urinary proteins, purification was achieved totally on the basis of size (see Fig. 1). Purity of the final product was estimated to be greater than 95%, based on SDS-polyacrylamide gel electrophoresis, amino acid composition and sequence analysis. The yield of /?2m varied between 10 and 15 mg per liter of urine. The complete amino acid sequence of guinea pig /?2m is depicted in Fig. 2. It is 99 residues long and has a molecular weight of 11,500 daltons by electrophoresis in SDS. The molecule contains two cysteinyl residues. Our purification requires the modification of these residues so that a demonstration of an intrachain disulfide bond could not be made. However, experiments of Cigen et al. (1978) suggest that a disulfide bridge does exist in guinea pig p2m. The sequence was determined as shown in Fig. 2. A traditional strategy was employed in determining the primary structure. Tryptic peptides which accounted for the entire sequence were isolated and characterized. Chymotrypsin was used to generate peptides with sequences overlapping the tryptic fragments. Trypsin was also used to generate large fragments by
GuineaPig /?ZMicroglobulin digesting fl2m which had been derivatized with thereby cleaving the succinic anhydride, molecule only after arginyl residues. This technique resulted in the generation of two important fragments, A3 and A4. Guinea pig fl2m has a single methionyl residue and cyanogen bromide was used to split the molecule into two fragments. Sequence information was obtained following this cleavage without fractionating the resulting peptides. Again, this was accomplished by using succinylated fl2m. With all primary amines blocked, reaction with cyanogen bromide generated a single new amino terminus and the products were directly subjected to sequence analysis. The most successful automated degradation of the intact molecule allowed the identification of 32 consecutive amino acid successful sequence deLess residues. terminations were usually terminated before position 32 by a substantial reduction in total yield following residue position 17 (asparagine). A partial sequence of guinea pig /?2m was reported which assigned glutamic acid (orglutamine) to residue position 6 (Cunningham et al., 1976). In all of our sequence analyses, including B2m from Hartley and strain 2 animals, arginine was found in the sixth position. Furthermore, no sequence heterogeneity was observed among microglobulins from any of these strains. An independent characterization of guinea pig /I2m describes the isolation of two forms of the molecule, a major form containing eight lysyl residues and a minor form containing only seven (Cigtn et al., 1978). Two explanations were offered to account for these variants: (a) they are products of distinct genes, or(b) the minor variety isaproteolyticproduct ofthemajorform. Ourdata support the latter alternative because digestion of guinea pig /?2m with carboxypeptidase B releases between 0.5 and 0.8 moles of lysine per mole of substrate. This indicates that 20-50x of the molecules express an amino acid residue other than lysine (or arginine) at the carboxyl 10 v
GUINEA PIG
RABBIT
HUMAN
20
30
V
V
1501
terminus. In addition,. the ctirboxyl-terminal tryptic peptide Tl 1, Trp-Asp-Pro-Asn-Lys, differs from Tl la only in that Tl la lacks a Cterminal lysyl residue. Both observations are consistent with the data of Cig&n et al. (1978). Thus it is likely that the minor form of guinea pig /?2m arises by the action of an exopeptidase on the major form. Additional support for proteolysis comes from a recent observation by Tucker et al. (1979). In determining the nucleotide sequence of mouse IgG2b heavy chain messenger RNA, they found a codon for a lysyl residue at the carboxyl-terminus of the molecule. This residue has not been found by amino acid sequence determinations of the mature protein. A serum protease was suggested to account for this post-translational modification. One might further hypothesize the occurrence of microglobulin molecules longer than 99 residues. If a proteolytic mechanism is involved in generating two forms of guinea pig /?2m, it may have initially operated on a substrate a few residues, longer than the molecules in urine. However, such an extension must be below the limits of resolution of molecular weight analyses, such as SDS-polyacrylamide gel electrophoresis, because no size variants have been detected by these methods. The real nature of the carboxyl terminus of guinea pig /?2m on cell surfaces still remains in doubt. Experiments designed to determine the carboxyl terminal residue of cell surface /?2m are currently in progress and these may help to establish the carboxyl-terminus of’ the functional form of the molecule. The complete primary structures of 82rn from human, rabbit and guinea pig have now been determined. A comparison of these sequences is presented in Fig. 7. The guinea pig molecule is identical to human /?2m at 68 positions and to rabbit B2m at 75 positions. Rabbit and human B2m share 72 residues. If amino acid substitutions which do not alter the charge or polarity of the side chains are considered equivalent (e.g. Leu/Ile at position 23 or Lys/Arg
40
50
V
V
60
70
V
V
80
90
99
V
V
V
VLHAPRV~VYSRHPAENGKDNFINCYVSGFHPPqIEVELLKNGKKIONVEMSDLSFSKO~FYLLVHAAFTPNDSDEYSCRVSHITLSEPKIVKWDPNK
--QR-N
P-L
IQRT-KI
K-V-K-MT-RDY
DI-V-E--Q-N-S-TE-NKN-
S-L-SD-D-D-ER-EK-H
-S-YYTE-TEK-A-N-V-_P-RDM
Fig.7. A comparisonof the primarystructuresof guineapig,rabbitandhuman line indicates
residues which are identical
to the guinea pig molecule. omitted (see discussion).
Residue
B2-microglobulin. The solid 67 of human fi2rn has been
1502
PAUL
B. WOLFE
at position 45) guinea pig fi2rn is homologous to human fi2m at 80 positions and to rabbit p2m at 82 positions. Human and rabbit fi2rn are homologous at 79 positions. Interpretations of such comparisons of primary structure are limited. Certainly this kind of comparison highlights the strong degree of structural similarity across wide species differences and suggests that the role of microglobulin is critical enough to warrant a high level of sequence conservation in evolution. A comparison of tertiary conformations is, of course, impossible because no information concerning the threedimensional structure is available. But the striking homology between b2m and the constant regions of immunoglobulins suggests a possible three-dimensional structure for microglobulins. Sequence comparisons (Cunningham, 1974) CD and ORD analyses (Karlsson, 1974; Isenman et al., 1975), the arrangement of cysteinyl residues into an intrachain disulfide bond (Cunningham et al., 1973) and preliminary X-ray diffraction analysis (Becker et al., 1977) all support the concept that B2m has three-dimensional features similar to the basic immunoglobulin fold (Poljak ei al., 1973). Beale & Feinstein (1976) have
r
SER
GLY
PHE
HIS
PRo@@7
2 “IS
TRP ASP
THR -II
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A!
iR
ME 00
I
ALA
PRO
ILE
A
TYR
GLN
ARG
GLU
CYS
“AL
CYS
"AL
ASN
TYR
SER
GLU
LYS
ALA
ALA
GLU "AL ASN ASP
ILE LYS LYS GLY AS 00
LYS LEU-
Fig. 8. A two-dimensional representation of the proposed three-dimensional structure of guinea pig /32-microglobulin, The boxed areas delineate regions of predicted fl-structure. Circled residues are those found to be non homologous among guinea pig, rabbit and human /32m. Strands of b-sheet (Sl, S2, S7) are numbered in order of their appearance from the amino- to the carboxyl-terminal end.
and JOHN
J. CEBRA
summarized several properties of the basic polypeptide chain fold of constant region domains based on a comparison of immunoglobulin domain sequences and the available crystallographic models. Using these as a guide, we have constructed a model of the three-dimensional structure of guinea pig p2m (Fig. 8) in the two-dimensional format of Saul et al. (1978). The model incorporates the following features of a constant homology region: (1) The intrachain disulfide bond is shielded by the tryptophyl residue at position 95. This residue is not strictly homologous to the invariant tryptophan which occurs in the domains of immunoglobulins. The tryptophan of /?2m is located in the S7 strand (nomenclature of Segal et al., 1974) whereas the tryptophan of constant homology regions is located in the S3 strand. the apparent importance of a However, tryptophandisulfide interaction in immunoglobulins prompted us to include this (2) Invariant arrangement in the model; hydrophobic residues, considered to be important in forming internal clusters which regulate domain folding, have been taken into consideration in assigning the orientation of residues in stretches of /?-structure; (3) Segments of alternating hydrophobic residues have been arranged such that the maximum amount of bstructure is symmetrically distributed around the disulfide bridge. The limits of this secondary structure are defined by the first and last residues in each segment bearing a side chain which is likely to be oriented toward the interior of the molecule. An alternating series of hydrophobic residues, although indicative of /?-structure, is never strictly observed. However, we have set the limits of P-sheets to include only this arrangement. This conservative assignment of residues into b-sheets does not significantly change the conclusions drawn from the model. Segments that bear proline and/or glycine (residues commonly found in regions where a discontinuity between areas of regular structure is required) and that do not contain a significant proportion of amino acids with hydrophobic side chains have been relegated to the bends connecting regions of p-structure. Amino- and carboxyl-terminal segments that contain proline are also excluded from these regions. In comparing microglobulin sequences we have adopted a convention designed to emphasize major structural differences. As we distinguish between mentioned earlier, identical and homologous residues by
Guinea Pig fiZMicroglobulin
considering interchanges which preserve the charge and polarity of the side chains as For the purpose of this homologous. comparison, the following groups of amino acids are taken to be equivalent: (a) Lys, His and Arg; (b) Thr, Ser and Ala; (c) Asp and Glu; (d) Asn and Gln; and (e) Val, Met, Ile, Leu, Tyr and Phe. This is not an unreasonable assumption as values from Grantham’s index (Grantham, 1974) a method for evaluating the average dissimilarity between any two amino acid residues, fall between 5 and 55 for these substitutions. The range of the index is from 5, for the dissimilarity between leucine and isoleucine, to 215, for dissimilarity between cysteine and tryptophan. When comparing sequences, differences are scored regardless of whether they occur between guinea pig and human, guinea pig and rabbit, or human and rabbit molecules. The value of examining microglobulins in this manner is that amino acid substitutions can be evaluated with respect to where they may occur in the tertiary structure. An inspection of strictly non-identical residues shows that 10 are found in the 37 residues assigned to P-sheets and 29 are found outside these areas. That is, 27% of residues within /?-sheets are non-identical compared to 47% non-identities in loops and terminal strands. The differences are more striking when homologous residues are considered. In this case only 2 of 37 residues (5%) are significantly different in regions of /Istructure, while 22 of 62 residues (35%) remain different outside these segments (circled residues in Fig. 8). It seems that the major differences among microglobulins occur in areas which do not contribute to forming a compact globular domain. In regions of supposed P-sheet, both the residues bearing side chains which constitute the hydrophobic core of the molecule and adjacent residues, with their side chains exposed to the environment, are overwhelmingly conserved. If we turn from the differences among microglobulins to the similarities, the Sl, S2, S4 and S5 strands, and connecting loops, draw the most attention. The highest proportion of conserved residues (40/46) are found on this face of the molecule. The traditional view of sequence conservation has been to assign a high relative importance to invariant segments of molecules, as these are usually crucial to maintenance of structure or efficiency of function. Given the evidence that fi2m is shaped like an immunoglobulin domain, perhaps it interacts with histocompatibility antigens as antibody
1503
domains interact with each other. Constant region domains interact through the 4-chain layers of b-structure whereas variable region domains interact through their 3-chain layers (Edmundson et al., 1975). Since p2m is more closely related in structure to constant regions than variable regions, it might be predicted that /?2m associates with its heavy chain through its 4chain layer as well. The failure to find B2m dimers would seem to weigh against this, but the relatively high proportion of residues with hydrophobic side chains facing away from the molecule, including an invariant tryptophan at residue position 60 at the presumed apex of a hairpin turn, and the abundance of conserved residues occupying this face appear to favor this face as being the site of interaction with histocompatibility antigens. Finally, one residue in the sequence of human close scrutiny. B2m requires particularly Previous comparisons of microglobulins from rabbit and guinea pig with the human sequence have required that an insertion or gap be introduced around residue position 67 in order to maximize homology to the human molecule (Gates et al., 1979; Wolfe & Cebra, 1978). A deletion or addition mutation giving rise to this arrangement seems unlikely given that microglobulins are a very strongly conserved class of proteins. A close examination of the data supporting residue assignments in this region of human /?2m (Cunningham et al., 1973) has led us to suggest that residue 67 (serine) in the human molecule may not exist. We base our opinion on the following evidence: (1) TN9, the tryptic peptide from human B2rn containing this residue, was never isolated free of contamination from tryptic peptide TN5. The amino acid composition of TN9 gave a serine content of 1.5 mol per mol peptide which was rounded to 2.0 as the nearest integral number of residues per peptide. However, TN5 has three seryl residues. If it contaminated TN9 by as little as 15% it could account for the extra 0.5 serine found in the composition of TN9; (2) Sequence analysis of TN9 was carried out by the dansyl-Edman method. This technique is not quantitative. Assignment of residues depends on visualization of a dansylated derivative of an amino acid on thin layer chromatography. The serine in question was assigned to position 9 in peptide TN9. Peptide TN5 has a serine in position 9 as well. Furthermore, the failure to assign tyrosine to position 9 in peptide TN9 may have been due to its dismissal as ‘carry-over’ from the previous
PAUL B. WOLFE
1504
position, which is also tyrosine; (3) Serine 67 in human /I2rn is the only residue for which there is no second, independent confirmation of its assignment. That is, there are no overlapping peptides which. verify its placement. These considerations cause us to suggest that the assignment of serine to position 67 may not be this residue from justified and to omit consideration in Fig. 7. Acknowledgements-We
wish to thank Ms. J. Lilly for expert technical assistance and Dr. R. F. Brunhouse for invaluable discussions and criticism of the manuscript, This work was supported by grants from the National Science Foundation (GB-38798) and the National Institute of Allergy and Infectious Diseases (AIq9652). P.B.W. has been supported as a pre-doctoral trainee by a training grant from the National Institutes of Health (GM00057).
REFERENCES Beale D. & Feinstein A. (1976) Structure and function of the constant regions of immunoglobulins. Q. Rev. Biophys. 9, 135-180. Becker J. W., Ziffer J. A., Edelman G. M. & Cunningham B. A. (1977) Crystallographic studies of bovine /I2microglobulin. Proc. n&n. Acad. Sci. U.S.A. 14, 3345-3349.
Berggard I. (1974) Isolation and characteristics of a rabbit b2-microglobulin: comparison with human 82microglobulin. Biochim. biophys. Res. Commun. 57, 1159-1165.
Berggard I. & Bearn A. G. (1968) Isolation and properties of a low molecular weight /?2-globulin occurring in human biological fluids. J. biol. Chem. 243, 49054103. Birshtein B. K., Turner K. J. & Cebra J. J. (1971) Structure of heavy chain from strain 13 guinea pig immunoglobulinG(2). I. Isolation of cyanogen bromide fragments. Biochemistry 10, l-8. Cebra J., Brunhouse R., Cordle C., Daiss J., Fechheimer M., Ricardo M., Thunberg A. & Wolfe P. B. (1977) Isotypes of guinea pig antibodies: restricted expression and bases for interactions with other molecules.Prog. Immun. 3, 269-277. C&en R., Ziffer J. A., Berggard B., Cunningham B. A. & Bernaard I. (1978) Guinea pig b2-microglobulin. Purification and partial structure. Biochemistr): 17, 947-955. B. A. (1974) B2-microglobulin: an Cunningham immunoglobulin domain associated with cell surfaces. Prog. Immun.
2, 5-12.
Cunningham B. A., Henning R., Milner R. J., Reske K., Ziffer J. A. & Edelman G. M. (1976) Structure of murine histocompatibility antigens. Cold Spring Hub. Symp. quant. Biol. 41, 351-362.
Cunningham B. A., Wang J. L., Berggard I. & Peterson P. A. (1973) The complete amino acid sequence of fi2microglobulin. Biochemistry 12, 48 I 111822. Doherty P. C., Blanden R. V. & Zinkernagel R. M. (1976) Specificity of virus-immune effector T cells for H-2K or H-2D compatible interactions: implications for H-antigen diversity. Transplantn. Rev. 29, 89-124. Easley C. W. (1965) Combinations of specific color reactions useful in the peptide mapping technique. Biochem. biophys. Acta 107, 36X-388.
Edmundson A. B., Ely K. R., Abola E. E., Schiffer M. & Panagiotopoulos N. (1975) Rotational allomerism and divergent evolution of domains in immunoglobulin light chains. Bi0chemistr.y 14, 3953-3961.
and JOHN J. CEBRA Fairbanks G., Steck T. L. & Wallach D. F. H. (1971) Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistr,, 10, 2606-26
16.
Fellous M., Gunther E., Kemler R., Wiels 3.. Berger R.. Guenot J. L.. Jakob H. & Jacob F. (1978) Association of the H-Y male antigen with /(2-microglobulin on human lymphoid and differentiated mouse teratocarcinoma cell lines. J. exp. Med. 148, 58-70. Gates F. T., Coligan J. E. & Kindt T. J. (1979) Complete amino acid sequence of rabbit fl2-microglobulin. Biochemistry
18, 2267-2272.
Grantham R. (1974)Amino acid difference formula to help explain protein evolution. Science 185, 862-864. Grey H. M., Kubo R. T., Colon S. M., Poulik M. D., Cresswell P., Springer T., Turner M. & Strominger J. L. (1973) The small subunit of HL-A antigens is 82microglobulin. J. up. Med. 138, 1608-1612. Hayashi R., Moore S. & Stein W. H. (1973) Carboxypeptidase from yeast. Large scale preparation and the application to COOH-terminal analysis of peptides and proteins. J. hiol. Chrm. 248, 2296-2302. Helenius A., Morein B., Fries E., Simons K., Robinson P., Schirrmacher V.. Terhorst C. & Strominger J. L. (1978) Human (HLA-A and HLA-B) and murine (H-2K and H-2D) histocompatibility antigens are cell surface receptors for Semliki Forest Virus. Proc. natn. Acad. Sci. U.S.A.
75, 3846-3850.
Isenman D. E., Painter R. H. & Dorrington K. J. (1975) The structure and function of immunoglobulin domains: studies with beta 2-microglobulin on the role of the intrachain disulfide bond. Proc. natn. Acad. Sci. U.S.A. 72, 548-552.
Karlsson F. A. (1974) Physical-chemical properties of 82. microglobulin. Immunochemistry 11, I1 l-l 14. Klotz I. M. (1967) Succinylation. Mefh. Enzym. 11,576-580. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, Lond.
227, 680-685.
Michaelson J., Flaherty L.. Vitetta E. & Poulik M. D. (1977) Molecular similarities between the Qa-2 alloantigen and other gene producis of the 17th chromosome of the mouse. J. exp. Med. 145, 1066-1070. Ostberg L., Rask L., Wigzell H. & Peterson P. A. (1975) Thymus leukaemia antigen contains fl2-microglobulin. Nature, Lond. 253, 735-737. Peterson P. A., Cunningham B. A., Berggard 1. & Edelman G. M. (1972) /?2-microglobulin: a free immunoglobulin domain. Proc. natn. Acad. Sri. U.S.A 69, 1697-1701. Peterson P. A., Rask L. & Lindblom J. B. (1974) Highly purified papain-solubilized HL-A antigens contain /I2microglobulin. Proc. natn. Acad. Sci. U.S.A. 71, 35-39. Poljak R. J., Amzel L. M., Avey H. P., Chen B. L.. Phizackerley R. P. & Saul F. (1973) Three-dimensional structure of the Fab’ fragment of a human immunoglobulin at 2.8-A resolution. Proc. natn. Acad. Sci. U.S.A.
70, 3305-3310.
Pouhk M. D., Shinnick C. J. & Smithies 0. (1975) Partial amino acid sequence of rabbit and rat /r’2-microglobulin. Fedn.
Proc.
34, 945.
Rask L., Lindblom J. B. & Peterson P. A. (1974) Subunit structure of H-2 alloantigens. Nature, Land. 249,833-834. Saul F. A.. Amzel L. M. & Poljak R. J. (1978) Preliminary refinement and structural analysis of the Fab fragment from human immunoglobulin New at 2.0 A resolution. J. hiol. Chem. 253, 585-597. Schwartz B. D., Cigen R.. Berggard I. & Shevach E. M. (1978) Guinea pig homologues of TL and QA-2 antigens. j. Zmmun. 121,.815-839.
-
Schwatz B. D.. Kask A. M.. Paul W. E. & Shevach E. M. (1976) Structural characteristics of the alloantigens determined by the major histocompatibility complex of the guinea pig. J. exp. Med. 143, 541-558.
Guinea
Pig b2-Microglobulin
Segal D. M., Padlan E. A. Cohen G. H., Rudikoff S., Potter M. & Davies D. R. (1974) The three-dimensional structure of a phosphorylcholine-binding mouse immunoglobulin Fab and the nature of the antigen binding site. Proc. natn. Acad. Sci. U.S.A. 71,4298-4302. Silver J. & Hood L. (1974) Detergent-solubilised H-2 alloantigen is associated with a small molecular weight polypeptide. Nature, Lond. 249, 764-765. Smithies 0. & Poulik M. D. (1972a) Initiation of protein synthesis at an unusual position in an immunoglobulin gene? Science 175, 187-189. Smithies 0. & Poulik M. D. (1972b) Dog homologue of human p2-microglobulin. Proc. natn. Acad. Sci. U.S.A. 69, 2914-2917. Summers M. R., Smythers G. W. & Oroszlan S. (1973) Thinlayer chromatography of sub-nanomole amounts of phenylthiohydantoin (PTH) amino acids on polyamide sheets. Analyt. Biochem. 53, 624-628. Tucker P. W., Marcu K. B., Slightom J. L. & Blattner F. R. (1979) Structure of the constant and 3’ untranslated regions of the murine y2b heavy chain messenger RNA. Science 206, 1299-1303.
1505
Turner K. J. & Cebra J. J. (1971) Structure of heavy chain from strain 13 guinea pig immunoglobulin-G(2). II. Amino acid sequence of the carboxyl-terminal and hinge region cyanogen bromide fragments. Biochemistry 10, 9-17 Vitetta E. S., Poulik M. D., Klein J. & Uhr J. W. (1976) Beta 2-microglobulin is selectively associated with H-2 and TL alloantigens on murine lymphoid cells. J. exp. Med. 144, 179-192. Vitetta E. S., Uhr J. W. & Boyse E. A. (1975) Association ofa /?2-microglobulin-like subunit with H-2 and TL alloantigens on murine thymocytes. J. Immun. 114, 252-254. Winkler M. A. & Sanders B. G. (1977) Chemical and immunologic characterization of a p2-microglobulin-like protein isolated from chicken sera. Immunochemistry. 14, 615619. Wolfe P. B. & Cebra J. J. (1978) Guinea pig 82microglobulin. Fed. Proc. 37, 1468. Yamada S. & Itano H. A. (1966) Phenanthrenequinone as an analytical reagent for arginine and other monosubstituted guanidines. Biochim. biophys. Acta 130, 538-540.
Note added in proof: The following was inadvertently omitted from the original manuscript: In order to obtain a peptide overlapping peptides T4 through T7, peptide A3 was digested with chymotrypsin. The A3 chymotryptic peptides were fractionated by gel filtration (not shown) which yielded a peptide extending from position 27 (Val) to position 56 (Phe) and was designated A3-Ch2. Automated sequential degradation of A3-Ch2 established the order of tryptic peptides T4 through T7 (Fig. 2).
IrnrnU~~l~Y, Press Ltd.
Vol. I?, pp. 1493-1505. 1980. Printed in Great
0161-SS#j8O~~ZOl-i493
102.00/O
Britain
THE PRIMARY STRUCTURE OF GUINEA PIG P2-MICROGLOBULIN PAUL B. WOLFE and JOHN J. CEBRA Department of Biology, The Johns Hopkins University, Baltimore, MD 21218, U.S.A. (Received 3 April 1980)
Abstract-The complete primary structure of guinea pig b2-microglobulin has been determined. It is 99 residues long and shares approximately 70% of its sequence with human and rabbit p2m. Heterogeneity at the carboxyl-terminus gives rise to two forms of the molecule: one bearing a COOH-terminal lysyl residue and one which lacks this residue. Evidence suggests that the two forms arise due to proteolysis, but multiple forms offl2m are not ruled out. A model of the three-dimensional structure is presented and used to evaluate amino acid substitutions among microglobulins. A high degree of conservation is seen among residues responsible for maintenance of fl-structure. This is especially true for residues occurring in a presumptive four-chain layer and suggests that this is the face which interacts with histocompatibility antigens.
INTRODUCTION
Interest in 82-microglobulin (/?2m) has stemmed from two general observations. It has clear structural homology to the constant regions of immunoglobulins (Smithies & Poulik, 1972a; Peterson et al., 1972). This similarity has prompted consideration of a close evolutionary relationship between these molecules (Smithies & Poulik, 1972a). The obse~ation of more general interest has been that B2m is a subunit of both major and minor histocompatibility antigens in the cell membrane. Human (HLA), mouse (H-2K and H-2D) and guinea pig (GPLA) major histocompatibility antigens (Grey et al., 1973; Peterson et al., 1974; Silver & Hood, 1974; Rask et al., 1974; Schwartz et al., 1976), as well as the minor histocompatibility antigens of the mouse (Tla, Qa-2 and H-Y) and their analogs in the guinea pig (Ostberg et al., 1975; Vitetta et al., 1975; Vitetta et al., 1976; Fellous et al., 1978; Michaelson et al., 1977; Schwartz et al., 1978) are paired with p2m. Thus, ,!32m may be a common subunit of all major transplantation antigens and structurally related cell surface molecules. The precise roles played by these highly polymorphic glycoproteins are unknown. Their appearance as differentiation antigens and virus receptors (Helenius et al., 1978) and their participation in T cell-mediated immunity (Doherty et al., 1976) suggest that they perform a variety of biological functions. In contrast, /32m remains the essentially invariant component, not only within this class of molecules, but across species lines as well.
Consequently, study of the structure and function of fi2rn may provide insight into the roles of its more complex partner. We herein describe the isolation of guinea pig ~2-mi~roglobulin and the determination of its primary structure. Microglobulins from a variety of species, including guinea pig, have been described (Smithies & Poulik, 1972b; Poulik et al., 1975; Becker et al., 1977; CigCn et al., 1978; Winkler & Sanders, 1977). The complete primary structures of human and rabbit p2m are published (Cunningham et al., 1973; Gates et al., 1979) and several partial sequences of this molecule from other species have appeared as well (Smithies & Poulik, 19726; Poulik e.! al., 1975; Becker et al., 1977; CigCn et al., 1978). These data establish the sequence homology of p2m with immunoglobulin constant region domains and demonstrate a high degree of structural similarity among microglobulins. Our results extend those observations by providing a third complete sequence for comparison. A preliminary report and partial sequence have appeared (Wolfe & Cebra, 1978; Cebra et al., 1977).
MATERIALS AND METHODS
Purification of guinea pig /?2m was based partly on procedures described for purification of human and rabbit p2m (Berggard & Bearn, 1968; Berggard, 1974). Six to twelve strain 2, strain 13 or Hartley guinea pigs, raised in our own colony, were given subcutaneous injections
1493
1494
PAUL
B. WOLFE
of potassium chromate (10 mg/kg) in saline. The animals were kept in metabolism cages (3-6 animals per cage) and urine was collected for seven days following administration of the poison. No deaths resulted directly from this treatment and animals were frequently used more than once after a suitable recovery period (3-4 weeks). Each 24-hr specimen was frozen immediately after collection into a container immersed in a dry ice/acetone bath. On average, six animals yielded one liter of urine over seven days. After collection, the urine was thawed and
and JOHN
J. CEBRA
centrifuged to remove insoluble material. The resulting supernatant was dialysed in the cold against several changes of phosphate-buffered saline at pH 7.3. Urinary proteins were concentrated and a crude fi2m-containing precipitate was obtained by the addition of cold, saturated ammonium sulfate to 60’/,, saturation. The suspension was stirred in the cold overnight, then allowed to settle so that the majority of the supernatant could be drawn off by siphon. The remainder was centrifuged to collect the precipitate, which was dissolved in a minimum volume, dialysed against IO mM Tris-HCl,
b
a 2.4 -
:
2.4 E = 20
20-
0 a,
0 :
N
16-
I6
w
:
ii
1.2-
a m
Z g
1.2
: m 0.8
0.8-
:
:
a 0.4-
I1
40
60
80
FRACTION
100 NUMBER
120
140
I6(
I
20
I
I
40
FRACTION
2’
III
1
60
I
80
I
I
I
100
I
120
NUMBER
d
FRACTION
NUMBER
Fig. 1. Purification of guinea pig /?2-microglobulin. (a) Concentrated urinary proteins fractionated on Sephadex G-100; (b) fraction 4 from G-100 on Sephadex G-50 in urea and formic acid; (c) fraction 2 from G-SO on Sephadex G-75 in urea and formic acid; (d) SDS-polyacrylamide gel electrophoresis: column 2, whole urinary proteins; column 3, G-100 fraction 4; column 4, G-50 fraction 2; column 5, G-75 fraction 2 columns 1 and 6, molecular weight standards. Lactoperoxidase = 77,500 daltons; ovalbumin = 43,500 daltons; trypsin = 23,800 daltons; and lysozyme = 12,000 daltons.
Guinea Pig j2-Microglobulin
pH 8.0, containing 1 M NaCl and 0.05% NaN,, and chromatographed on a column of Sephadex G-100 (3.2 x 180 cm) equilibrated in the same buffer. It was found that fraction 4 (Fig. la) contained material of the appropriate molecular weight as assessed by SDS-polyacrylamide gel electrophoresis (Fig. Id), by the elution volume of a radiolabeled sample of rabbit P2m (data not shown) and by comparison to published observations using this chromatography system (Berggard, 1974). The material in this fraction was pooled, desalted on a column of Sephadex G-50, coarse (3 x 48 cm), equilibrated in 50 mM formic acid, and lyophilized. The lyophilized material was dissolved in 8 M urea which was 0.1 M in formic acid and chromatographed on a column of Sephadex G-50, superfine (2.5 x 180 cm), equilibrated in the same solvent. Fraction 2 (Fig. lb) contained material of the appropriate molecular weight as judged by SDS-polyacrylamide gel electrophoresis. This pool was desalted and lyophilized as before, subjected to total reduction and alkylation with iodoacetic acid (Birshtein et al., 1971) and chromatographed on a column of Sephadex G-75 (2.5 x 180 cm) in 8 Murea, 0.1 Min formic acid. Fraction 2 (Fig. lc) contained material of 11,500 daltons without significant contamination (Fig. Id). Amino acid analysis and N-terminal sequence analysis both indicated the presence of a homogeneous product. Automated sequential degradation
Carboxymethylated fi2m and peptides derived from it were submitted to automatic sequential degradation in a Beckman 890-B sequencer. The peptide program (111374, Beckman Instruments Corp.), using dimethylallylamine as a solvent buffer, was employed for all sequence determinations. For peptides which were small and/or hydrophobic, 50 ,ug of polybrene (Aldrich Chemicals, Milwaukee, WI) was used as a carrier. Polybrene was subjected to two full cycles of Edman degradation prior to the addition of peptide. Identification of the amino acid residues at each position was accomplished by procedures previously described (Birshtein et al., 1971). PTH derivatives of amino acids were also identified by thin layer chromatography on polyamide sheets (Summers et al., 1973) and by calorimetric methods (Yamada & Itano, 1966; Easley, 1965). Enzymic digestion of p2m
Carboxymethylated
/?2m (l-l .5 pmol) was
1495
digested at 37°C in 0.5% ammonium bicarbonate by the addition of trypsin (Code: TRTPCK, Worthington Biochemical Corp; Freehold, NJ) at an enzyme to substrate ratio of 1:200 (w/w) at hourly intervals for 4 hr. The reaction was allowed to continue for an additional 4 hr at 37°C without further addition of the enzyme. Carboxymethylated /32m (0.5-l pmol) was digested with a-chymotrypsin (Worthington Biochemical Corp.) under conditions identical with those for trypsin digestion. Carboxypeptidase A and carboxypeptidase B were used as previously described (Turner & Cebra, 1971). Carboxypeptidase Y (Worthington Biochemical Corp.) was used according to Hayashi et al. (1973). Succinylation
Carboxymethylated /?2m (0.5 pmol) was succinylated using a modification of the method suggested by Klotz (1967). The protein was dissolved in 5 ml of 6 M guanidine, 0.1 M borate, pH 9.1. Solid succinic anhydride, lOO-fold molar excess over available amino groups, was added slowly with stirring at room temperature. The pH was maintained by dropwise addition of 1 N NaOH. The reaction was continued for 30 min after the last addition of anhydride. Excess reagents were removed by dialysis against 0.1 M formic acid and the product was lyophilized. The extent of derivatization was checked by digesting a small sample (2-5 nmol) with trypsin (4 hr at 37°C) followed by digestion with carboxypeptidase B (4 hr at 37°C). The amount of lysine released as determined by amino acid analysis was always less than 1% of that obtained after acid hydrolysis of an equal amount of underivatized /?2m. Cyanogen bromide cleavage
Succinylated b2m (0.3 ,umol) was dissolved in 2 ml of 70% formic acid and chilled to 4°C. Quantitative cleavage at the single methionyl residue was achieved by addition of solid cyanogen bromide, then allowing the mixture to stand at 4°C for 24 hr (Birshtein et al., 1971). The reaction was terminated by addition of 10 vol. water followed by lyophilization. Other methods
Preparative high voltage paper electrophoresis was performed according to Turner & Cebra (1971). SDS-polyacrylamide gel electrophoresis was carried out as described by Laemmli (1970).
1496
PAUL 10
B. WOLFE
and JOHN
J. CEBRA 40
30
20
“LHAPR”PY~SRHPAENGK~~~*~~~“~~~~~~~~~~~~~~~~~~~~~“~ .____________________~~~~~~~~~~~~ T2 TI T3 T4 . --_~-_---r-_* ____________________----------
50
T5
--_
+GP T7 ;e T6-T7
4_-d-d Chl
.
c
Ch4
Ch3
-.
*
A3~~,,~~~~~~~~~~~~~~~~~~~~
Ch 2
A3
-
T6-T7-T6o
T8b
TS
T7
_
__
_
__
_
TII
TIO
___________
-
L-m Tllo
-D
T6-T7
_ v-_-4
Ch6
Ch7
Ch6 -e-d___
__-a__-
ChS
a__
Ch IO ________
Ch12
CNBr
.
_____4________
_---___
l
A3
_-.____--_-_______
A4
Fig. 2. A schematic representation of the procedure and resulting data used to deduce the sequence of guinea pig P2-microglobulin. Symbols used are: (+), removal of a residue by automatic Edman degradation; (-), identification of a residue by digestion with carboxypeptidase A; (-), identification of a residue by digestion with carboxypeptidase B.
Staining
procedures
were those of Fairbanks
et
al. (1971). RESULTS
Amino
terminal sequence of p2m
Automated sequential degradation of carboxymethylated guinea pig p2m (0.5 pmol) permitted identification of amino acids at 32 consecutive positions (Fig. 2). No sequence heterogeneity was observed among the first 25 residues in microglobulins obtained from Hartley, strain 2 or strain 13 animals. Carboxypeptidase
digestion of P2m
Digestion of carboxymethylated p2m with carboxypeptidases A, B or Y under nondenaturing conditions failed to release amino acids above control levels observed in the absence of substrate. Digestion with carboxypeptidase Y (CPY) in the presence of 6 M urea also failed to release carboxyl-terminal amino acids. However, a contaminating endopeptidase
activity in the CPY quantitatively split p2m between residues 65 and 66, which allowed the CPY to act efficiently at this site. Digestion with carboxypeptidase B (CPB) in the presence of 0.1% sodium dodecyl sulfate released from 0.5 to 0.8 moles of lysine per mole of substrate, depending on the preparation of 82m. Tryptic peptides Digestion of carboxymethylated b2m with trypsin and subsequent fractionation of tryptic peptides resulted in the isolation of a set of peptides which accounted for its entire amino acid sequence. Peptides Tl-T5, T7, and T9-Tl l were isolated as conventional tryptic fragments. Two partial digest products and one unexpected cleavage site resulted in the isolation of several related tryptic fragments. Peptide T6 is free lysine, which was not isolated. However, a variant of T7 (named T6-T7) was recovered with a lysyl residue at its amino-terminus. Peptide T8 was never isolated intact. Instead, peptides T8a and T8b, resulting from a chymotryptic-like
Guinea Pig /32-Microglobulin T6b T6-T7-T6a I
T4 I
T3 t
T9 t
T2 t
1497
TI t
TIO
1 56
2.8
2.4
d
0
0.4
60
00
100
120
FRACTION
140
160
160
200
NUMBER
Fig. 3. Gel filtration of tryptic peptides of fl2-microglobulin on a column of Sephadex G-50, superfine (2.5 x lSOcm), equilibrated in 50 mMammonium hydroxide. The solid line indicates absorbance at 215 nm. The dashed line indicates radioactivity originating from [3H]-carboxymethyl-cysteinyl residues. Fraction volume is 5.0 ml.
T7 ‘1
T4+Tllo
I
40
T6-T7
I
60 FRACTION
81,
I
80
100
T9 T3 T5
,
I
120
I
(
140
NUMBER
Fig. 4. Ion-exchange chromatography of tryptic peptides of /I2-microglobulin soluble at pH 3.1 on a column of sulfonated polystyrene (Beckman PA-35). The column was operated at 56°C with a linear 300 ml gradient of pyridinium acetate buffers, W-200 mM in pyridine, pH 3.1-5.4, which were 5% in I-propanol. The solid line indicates the relative fluorescence monitored at 480 nm following reaction with fluorescamine.
PAUL
1498
B. WOLFE
and JOHN
cleavage, were always observed. Peptide T8a was recovered as T6-T7-T8a. Apparently trypsin did not quantitatively cleave the lysyl-isoleucyl bond joining T6 and T7 or the lysyl-aspartyl bond linking T7 and T8a. Finally, a variant of the carboxyl-terminal peptide, Tl 1, was isolated. Peptide Tl la differs from Tl 1 only in that it lacks a C-terminal lysyl residue. Degradation prior to the isolation of the intact molecule is a plausible explanation for the appearance of this peptide. The tryptic digest was initially fractionated on a column of Sephadex G-50 as illustrated in Fig. 3. Pool 1 yielded peptides T6-T7-T8a and T8b after additional fractionation by gel chromatography (data not shown). Pools 2 and 3 contained peptides T4 and T3, respectively, and did not require further treatment. The other peptides eluted from this column were purified by high voltage paper el~trophoresis. Peptide T9 was isolated by this method from pool 4, T2 from pool 5, Tl from pool 6 and TlO from pool 7. Tryptic peptides soluble at pH 3.1 were also fractionated by ion-exchange chromatography. Figure 4 shows the results of fractionation on a sulfonated polystyrene resin (PA-35, Beckman Instruments Corp.). Peptides T3, T5, T6-T7, T7 and Tll were isolated directly by this method from pools 10, 11, 7,2 and 9, respectively. Pool 4 contained an equimolar mix of T4 and Tl 1a. Gel filtration of this mixture on Sephadex G-25, superfine (2 x 35 cm), in 60 mA4 HCl resolved these two peptides. Sequences and partial sequences of some of the tryptic peptides were determined as shown in
Table Tl LYS His Arg CMCys Asp Thr Ser Glu Pro GUY Ala Val Met Ile Leu Tyr Phe Trp
0.7 0.8
T2
1. Amino
Fig. 2. The amino acid compositions of all of the tryptic peptides are shown in Table 1. Chymotryptic
of the tryptic
peptides T6-T7-T8a
T4
T5
T&T7
T7
1.1 0.9
1.0 0.9
1.2
1.9
1.1
1.1
A:f
0.9 1.0 1.3
2.0
1.0 0.9
1.1 0.9 1.0 0.9
n
1.0 3.9 2.0 1.0 2.1 1.9 2.1 1.0 1.8
2.1 2.3 1.3
2.9 2.3 2.2
from ~2-microglobulin TSb
1.7
1.1
1.0
peptides
Peptides from a ~hymotryptic digest of carboxymethylated /?2m were fractionated by ion-exchange chromatography on a column of Beckman PA-35 as shown in Fig. 5. Pool 1 contained an assortment of fragments which eventually yielded peptide Ch9 after additional resolution by gel filtration (data not shown). Peptides Ch3 and Ch4 were resolved after chromatography of pool 2 on a column of Sephadex G-25, superfine (2 x 35 cm), equilibrated in 60 mA4 HCl. Pools 3 and 4 contained peptides Ch12 and Ch7, respectively, and required no further purification. Pool 7 yielded peptides Ch6 and ChlO after fractionation on a column of Sephadex G-25 (2.5 x 180 cm) in 50 mM ammonium bicarbonate which had been adjusted to pH 10 with ammonium hydroxide. Peptides in pools 6 and 8 were purified by preparative high voltage paper electrophoresis. Peptide Ch8 was isolated from pool 6, peptide Chl from pool 8. The amino acid compositions of the chymotryptic peptides are shown in Table 2. of sequence analysis of The results chymotryptic peptides are summarized in Fig. 2. Peptide Chl verifies an overlap between tryptic peptides Tl and T2, peptide Ch6 confirms the overlap between T7 and T8a, and peptide ChlO establishes the overlap between T8b and T9. Peptides Ch8 and Ch9 together confirm the sequence of T8b.
T3
1
1.0 1.1
acid compositions
J. CEBRA
4.2 1.0 2.6 1.5
0.9 1.0 1.3 3.7 1.1 2.1 1.4 1.2
T9
T10
1.1 0.7
0.9
0.9 1.7 1.1 1.3
Tlla
Tll 0.9
1.9
2.0
1.0
0.9
i-
+
0.9 1.1 0.8 0.9 1.2
1.3 0.6 1.2 1.4
0.9
1.3
1.2 0.5
2.0 1.2
I .o 1.2 I.0 I .8 +
2.3 1.1 1.2
1.0
1.1
0.9 1.0
1.0
Guinea Pig /72-Microglobulin ch9
,t
0112Ch7
Ch3+Ch4
t
t
Chg Ch6+ChlOCh I
t
t t
,,,,,
,
I
,
20
I
1499
56
1
Jl,
I,
I
40
60 FRACTION
00
100
6 *
120
140
NUMBER
Fig. 5. Ion-exchange chromatography of chymotryptic peptides of /?2-microglobulin on a column of sulfonated polystyrene (Beckman PA-35). The column was operated at 56°C with a linear 300 ml gradient of pyridinium acetate buffers, SO-200 mMin pyridine, pH 3.1-5.4, which were 5% in 1-propanol. The solid line indicates the relative fluorescence monitored at 480 nm following reaction with fluorescamine.
Sequence following cyanogen bromide cleavage
Carboxymethylated /32m was treated with succinic anhydride to block all primary amines, then cleaved with cyanogen bromide. Cleavage after the single methionyl residue generated a new amino terminus and the products of the reaction were directly subjected to automated sequence analysis without further treatment. Results of this analysis are shown in Fig. 2. The sequence
begins at Ser-52 and by necessity assigns the methionine to residue position 51. It also confirms the carboxyl-terminal sequence of tryptic peptide T7 and establishes an overlap across T7, T8a and T8b. Succinylated tryptic and chymotryptic peptides
Succinylated /?2m was digested with trypsin in order to obtain peptides resulting from cleavage
Table 2. Amino acid compositions of the chymotryptic peptides from /32-microglobulin Chl
Ch3
Ch4
Ch7
Ch8
Ch9
ChlO
1.2
LYS
His Arg CMCys Asp Thr Ser Glu Pro Gly Ala Val Met Ile Leu Tyr Phe Trp
Ch6
0.8 1.0
1.0
1.0 1.0 1.0
1.1 0.9
0.9 3.1 2.2 1.1
1.5
1.0 0.9 0.8
1.7 0.9 1.4 1.0
2.1
2.1 1.1
1.1
0.8 2.1
2.0
0.9 1.0
0.9
2.2
0.8 1.0 1.0 3.1 1.0 0.9 1.1 1.3
1.0 1.0 1.0
1.0 1.2
0.8 +
Ch12
1.0 1.0
1.0 1.1
1500
PAUL
B. WOLFE
400
and JOHN
J. CEBRA
600 EFFLUENT
VOLUME
(m/s)
Fig. 6. Gel filtration of tryptic peptides of succinylated P2-microglobulin on a column of Sephadex G-50, superfine (2.5 x 180 cm), equilibrated in 50 mM ammonium bicarbonate adjusted to pH 10.0 with ammonium hydroxide. The solid line indicates the absorbance at 280 nm. The dashed line indicates radioactivity originating from [3H]-carboxymethylcysteinyl residues. Fraction volume is 5.0 ml.
after arginyl residues. These peptides were fractionated on a column of Sephadex G-SO as shown in Fig. 6. Material in pool 1 was found to correspond in amino acid composition to the peptide extending from His-13 to Arg-81 and was designated peptide A3. Apparently trypsin failed to cleave the succinylated molecule between T8a and T8b as it did the underivatized protein. Pool 2 contained the peptide designated A4 which extends from Val-82 to the carboxylterminal end. Automated sequential degradation of A4 resulted in the determination of its complete sequence which established the order of tryptic peptides T9, TlO and Tl 1. As expected, pool 3 contained a mixture of the N-terminal hexapeptides Al and A2 (Tl and T2). DISCUSSION
Our initial approach to the purification of guinea pig P2-microglobulin was based on procedures described for the isolation of human and rabbit /I2rn (Berggard & Bearn, 1968; Berggard, 1974). These methods employ ionexchange or electrophoretic techniques in isolating this protein. We found methods involving separation by net charge to be impractical due to the presence of a contaminant which was yellow-brown in color and conferred charge heterogeneity to most of the urinary proteins. The contaminant remained associated with /?2m in the presence of urea and formic acid.
A suspected covalent association was confirmed when, after reduction and alkylation of cysteinyl residues, the material was easily removed by dialysis. No attempts were made to further characterize the contaminating material. With the exception of ammonium sulfate precipitation, used to concentrate urinary proteins, purification was achieved totally on the basis of size (see Fig. 1). Purity of the final product was estimated to be greater than 95%, based on SDS-polyacrylamide gel electrophoresis, amino acid composition and sequence analysis. The yield of /?2m varied between 10 and 15 mg per liter of urine. The complete amino acid sequence of guinea pig /?2m is depicted in Fig. 2. It is 99 residues long and has a molecular weight of 11,500 daltons by electrophoresis in SDS. The molecule contains two cysteinyl residues. Our purification requires the modification of these residues so that a demonstration of an intrachain disulfide bond could not be made. However, experiments of Cigen et al. (1978) suggest that a disulfide bridge does exist in guinea pig p2m. The sequence was determined as shown in Fig. 2. A traditional strategy was employed in determining the primary structure. Tryptic peptides which accounted for the entire sequence were isolated and characterized. Chymotrypsin was used to generate peptides with sequences overlapping the tryptic fragments. Trypsin was also used to generate large fragments by
GuineaPig /?ZMicroglobulin digesting fl2m which had been derivatized with thereby cleaving the succinic anhydride, molecule only after arginyl residues. This technique resulted in the generation of two important fragments, A3 and A4. Guinea pig fl2m has a single methionyl residue and cyanogen bromide was used to split the molecule into two fragments. Sequence information was obtained following this cleavage without fractionating the resulting peptides. Again, this was accomplished by using succinylated fl2m. With all primary amines blocked, reaction with cyanogen bromide generated a single new amino terminus and the products were directly subjected to sequence analysis. The most successful automated degradation of the intact molecule allowed the identification of 32 consecutive amino acid successful sequence deLess residues. terminations were usually terminated before position 32 by a substantial reduction in total yield following residue position 17 (asparagine). A partial sequence of guinea pig /?2m was reported which assigned glutamic acid (orglutamine) to residue position 6 (Cunningham et al., 1976). In all of our sequence analyses, including B2m from Hartley and strain 2 animals, arginine was found in the sixth position. Furthermore, no sequence heterogeneity was observed among microglobulins from any of these strains. An independent characterization of guinea pig /I2m describes the isolation of two forms of the molecule, a major form containing eight lysyl residues and a minor form containing only seven (Cigtn et al., 1978). Two explanations were offered to account for these variants: (a) they are products of distinct genes, or(b) the minor variety isaproteolyticproduct ofthemajorform. Ourdata support the latter alternative because digestion of guinea pig /?2m with carboxypeptidase B releases between 0.5 and 0.8 moles of lysine per mole of substrate. This indicates that 20-50x of the molecules express an amino acid residue other than lysine (or arginine) at the carboxyl 10 v
GUINEA PIG
RABBIT
HUMAN
20
30
V
V
1501
terminus. In addition,. the ctirboxyl-terminal tryptic peptide Tl 1, Trp-Asp-Pro-Asn-Lys, differs from Tl la only in that Tl la lacks a Cterminal lysyl residue. Both observations are consistent with the data of Cig&n et al. (1978). Thus it is likely that the minor form of guinea pig /?2m arises by the action of an exopeptidase on the major form. Additional support for proteolysis comes from a recent observation by Tucker et al. (1979). In determining the nucleotide sequence of mouse IgG2b heavy chain messenger RNA, they found a codon for a lysyl residue at the carboxyl-terminus of the molecule. This residue has not been found by amino acid sequence determinations of the mature protein. A serum protease was suggested to account for this post-translational modification. One might further hypothesize the occurrence of microglobulin molecules longer than 99 residues. If a proteolytic mechanism is involved in generating two forms of guinea pig /?2m, it may have initially operated on a substrate a few residues, longer than the molecules in urine. However, such an extension must be below the limits of resolution of molecular weight analyses, such as SDS-polyacrylamide gel electrophoresis, because no size variants have been detected by these methods. The real nature of the carboxyl terminus of guinea pig /?2m on cell surfaces still remains in doubt. Experiments designed to determine the carboxyl terminal residue of cell surface /?2m are currently in progress and these may help to establish the carboxyl-terminus of’ the functional form of the molecule. The complete primary structures of 82rn from human, rabbit and guinea pig have now been determined. A comparison of these sequences is presented in Fig. 7. The guinea pig molecule is identical to human /?2m at 68 positions and to rabbit B2m at 75 positions. Rabbit and human B2m share 72 residues. If amino acid substitutions which do not alter the charge or polarity of the side chains are considered equivalent (e.g. Leu/Ile at position 23 or Lys/Arg
40
50
V
V
60
70
V
V
80
90
99
V
V
V
VLHAPRV~VYSRHPAENGKDNFINCYVSGFHPPqIEVELLKNGKKIONVEMSDLSFSKO~FYLLVHAAFTPNDSDEYSCRVSHITLSEPKIVKWDPNK
--QR-N
P-L
IQRT-KI
K-V-K-MT-RDY
DI-V-E--Q-N-S-TE-NKN-
S-L-SD-D-D-ER-EK-H
-S-YYTE-TEK-A-N-V-_P-RDM
Fig.7. A comparisonof the primarystructuresof guineapig,rabbitandhuman line indicates
residues which are identical
to the guinea pig molecule. omitted (see discussion).
Residue
B2-microglobulin. The solid 67 of human fi2rn has been
1502
PAUL
B. WOLFE
at position 45) guinea pig fi2rn is homologous to human fi2m at 80 positions and to rabbit p2m at 82 positions. Human and rabbit fi2rn are homologous at 79 positions. Interpretations of such comparisons of primary structure are limited. Certainly this kind of comparison highlights the strong degree of structural similarity across wide species differences and suggests that the role of microglobulin is critical enough to warrant a high level of sequence conservation in evolution. A comparison of tertiary conformations is, of course, impossible because no information concerning the threedimensional structure is available. But the striking homology between b2m and the constant regions of immunoglobulins suggests a possible three-dimensional structure for microglobulins. Sequence comparisons (Cunningham, 1974) CD and ORD analyses (Karlsson, 1974; Isenman et al., 1975), the arrangement of cysteinyl residues into an intrachain disulfide bond (Cunningham et al., 1973) and preliminary X-ray diffraction analysis (Becker et al., 1977) all support the concept that B2m has three-dimensional features similar to the basic immunoglobulin fold (Poljak ei al., 1973). Beale & Feinstein (1976) have
r
SER
GLY
PHE
HIS
PRo@@7
2 “IS
TRP ASP
THR -II
3R
LEU
A!
iR
ME 00
I
ALA
PRO
ILE
A
TYR
GLN
ARG
GLU
CYS
“AL
CYS
"AL
ASN
TYR
SER
GLU
LYS
ALA
ALA
GLU "AL ASN ASP
ILE LYS LYS GLY AS 00
LYS LEU-
Fig. 8. A two-dimensional representation of the proposed three-dimensional structure of guinea pig /32-microglobulin, The boxed areas delineate regions of predicted fl-structure. Circled residues are those found to be non homologous among guinea pig, rabbit and human /32m. Strands of b-sheet (Sl, S2, S7) are numbered in order of their appearance from the amino- to the carboxyl-terminal end.
and JOHN
J. CEBRA
summarized several properties of the basic polypeptide chain fold of constant region domains based on a comparison of immunoglobulin domain sequences and the available crystallographic models. Using these as a guide, we have constructed a model of the three-dimensional structure of guinea pig p2m (Fig. 8) in the two-dimensional format of Saul et al. (1978). The model incorporates the following features of a constant homology region: (1) The intrachain disulfide bond is shielded by the tryptophyl residue at position 95. This residue is not strictly homologous to the invariant tryptophan which occurs in the domains of immunoglobulins. The tryptophan of /?2m is located in the S7 strand (nomenclature of Segal et al., 1974) whereas the tryptophan of constant homology regions is located in the S3 strand. the apparent importance of a However, tryptophandisulfide interaction in immunoglobulins prompted us to include this (2) Invariant arrangement in the model; hydrophobic residues, considered to be important in forming internal clusters which regulate domain folding, have been taken into consideration in assigning the orientation of residues in stretches of /?-structure; (3) Segments of alternating hydrophobic residues have been arranged such that the maximum amount of bstructure is symmetrically distributed around the disulfide bridge. The limits of this secondary structure are defined by the first and last residues in each segment bearing a side chain which is likely to be oriented toward the interior of the molecule. An alternating series of hydrophobic residues, although indicative of /?-structure, is never strictly observed. However, we have set the limits of P-sheets to include only this arrangement. This conservative assignment of residues into b-sheets does not significantly change the conclusions drawn from the model. Segments that bear proline and/or glycine (residues commonly found in regions where a discontinuity between areas of regular structure is required) and that do not contain a significant proportion of amino acids with hydrophobic side chains have been relegated to the bends connecting regions of p-structure. Amino- and carboxyl-terminal segments that contain proline are also excluded from these regions. In comparing microglobulin sequences we have adopted a convention designed to emphasize major structural differences. As we distinguish between mentioned earlier, identical and homologous residues by
Guinea Pig fiZMicroglobulin
considering interchanges which preserve the charge and polarity of the side chains as For the purpose of this homologous. comparison, the following groups of amino acids are taken to be equivalent: (a) Lys, His and Arg; (b) Thr, Ser and Ala; (c) Asp and Glu; (d) Asn and Gln; and (e) Val, Met, Ile, Leu, Tyr and Phe. This is not an unreasonable assumption as values from Grantham’s index (Grantham, 1974) a method for evaluating the average dissimilarity between any two amino acid residues, fall between 5 and 55 for these substitutions. The range of the index is from 5, for the dissimilarity between leucine and isoleucine, to 215, for dissimilarity between cysteine and tryptophan. When comparing sequences, differences are scored regardless of whether they occur between guinea pig and human, guinea pig and rabbit, or human and rabbit molecules. The value of examining microglobulins in this manner is that amino acid substitutions can be evaluated with respect to where they may occur in the tertiary structure. An inspection of strictly non-identical residues shows that 10 are found in the 37 residues assigned to P-sheets and 29 are found outside these areas. That is, 27% of residues within /?-sheets are non-identical compared to 47% non-identities in loops and terminal strands. The differences are more striking when homologous residues are considered. In this case only 2 of 37 residues (5%) are significantly different in regions of /Istructure, while 22 of 62 residues (35%) remain different outside these segments (circled residues in Fig. 8). It seems that the major differences among microglobulins occur in areas which do not contribute to forming a compact globular domain. In regions of supposed P-sheet, both the residues bearing side chains which constitute the hydrophobic core of the molecule and adjacent residues, with their side chains exposed to the environment, are overwhelmingly conserved. If we turn from the differences among microglobulins to the similarities, the Sl, S2, S4 and S5 strands, and connecting loops, draw the most attention. The highest proportion of conserved residues (40/46) are found on this face of the molecule. The traditional view of sequence conservation has been to assign a high relative importance to invariant segments of molecules, as these are usually crucial to maintenance of structure or efficiency of function. Given the evidence that fi2m is shaped like an immunoglobulin domain, perhaps it interacts with histocompatibility antigens as antibody
1503
domains interact with each other. Constant region domains interact through the 4-chain layers of b-structure whereas variable region domains interact through their 3-chain layers (Edmundson et al., 1975). Since p2m is more closely related in structure to constant regions than variable regions, it might be predicted that /?2m associates with its heavy chain through its 4chain layer as well. The failure to find B2m dimers would seem to weigh against this, but the relatively high proportion of residues with hydrophobic side chains facing away from the molecule, including an invariant tryptophan at residue position 60 at the presumed apex of a hairpin turn, and the abundance of conserved residues occupying this face appear to favor this face as being the site of interaction with histocompatibility antigens. Finally, one residue in the sequence of human close scrutiny. B2m requires particularly Previous comparisons of microglobulins from rabbit and guinea pig with the human sequence have required that an insertion or gap be introduced around residue position 67 in order to maximize homology to the human molecule (Gates et al., 1979; Wolfe & Cebra, 1978). A deletion or addition mutation giving rise to this arrangement seems unlikely given that microglobulins are a very strongly conserved class of proteins. A close examination of the data supporting residue assignments in this region of human /?2m (Cunningham et al., 1973) has led us to suggest that residue 67 (serine) in the human molecule may not exist. We base our opinion on the following evidence: (1) TN9, the tryptic peptide from human B2rn containing this residue, was never isolated free of contamination from tryptic peptide TN5. The amino acid composition of TN9 gave a serine content of 1.5 mol per mol peptide which was rounded to 2.0 as the nearest integral number of residues per peptide. However, TN5 has three seryl residues. If it contaminated TN9 by as little as 15% it could account for the extra 0.5 serine found in the composition of TN9; (2) Sequence analysis of TN9 was carried out by the dansyl-Edman method. This technique is not quantitative. Assignment of residues depends on visualization of a dansylated derivative of an amino acid on thin layer chromatography. The serine in question was assigned to position 9 in peptide TN9. Peptide TN5 has a serine in position 9 as well. Furthermore, the failure to assign tyrosine to position 9 in peptide TN9 may have been due to its dismissal as ‘carry-over’ from the previous
PAUL B. WOLFE
1504
position, which is also tyrosine; (3) Serine 67 in human /I2rn is the only residue for which there is no second, independent confirmation of its assignment. That is, there are no overlapping peptides which. verify its placement. These considerations cause us to suggest that the assignment of serine to position 67 may not be this residue from justified and to omit consideration in Fig. 7. Acknowledgements-We
wish to thank Ms. J. Lilly for expert technical assistance and Dr. R. F. Brunhouse for invaluable discussions and criticism of the manuscript, This work was supported by grants from the National Science Foundation (GB-38798) and the National Institute of Allergy and Infectious Diseases (AIq9652). P.B.W. has been supported as a pre-doctoral trainee by a training grant from the National Institutes of Health (GM00057).
REFERENCES Beale D. & Feinstein A. (1976) Structure and function of the constant regions of immunoglobulins. Q. Rev. Biophys. 9, 135-180. Becker J. W., Ziffer J. A., Edelman G. M. & Cunningham B. A. (1977) Crystallographic studies of bovine /I2microglobulin. Proc. n&n. Acad. Sci. U.S.A. 14, 3345-3349.
Berggard I. (1974) Isolation and characteristics of a rabbit b2-microglobulin: comparison with human 82microglobulin. Biochim. biophys. Res. Commun. 57, 1159-1165.
Berggard I. & Bearn A. G. (1968) Isolation and properties of a low molecular weight /?2-globulin occurring in human biological fluids. J. biol. Chem. 243, 49054103. Birshtein B. K., Turner K. J. & Cebra J. J. (1971) Structure of heavy chain from strain 13 guinea pig immunoglobulinG(2). I. Isolation of cyanogen bromide fragments. Biochemistry 10, l-8. Cebra J., Brunhouse R., Cordle C., Daiss J., Fechheimer M., Ricardo M., Thunberg A. & Wolfe P. B. (1977) Isotypes of guinea pig antibodies: restricted expression and bases for interactions with other molecules.Prog. Immun. 3, 269-277. C&en R., Ziffer J. A., Berggard B., Cunningham B. A. & Bernaard I. (1978) Guinea pig b2-microglobulin. Purification and partial structure. Biochemistr): 17, 947-955. B. A. (1974) B2-microglobulin: an Cunningham immunoglobulin domain associated with cell surfaces. Prog. Immun.
2, 5-12.
Cunningham B. A., Henning R., Milner R. J., Reske K., Ziffer J. A. & Edelman G. M. (1976) Structure of murine histocompatibility antigens. Cold Spring Hub. Symp. quant. Biol. 41, 351-362.
Cunningham B. A., Wang J. L., Berggard I. & Peterson P. A. (1973) The complete amino acid sequence of fi2microglobulin. Biochemistry 12, 48 I 111822. Doherty P. C., Blanden R. V. & Zinkernagel R. M. (1976) Specificity of virus-immune effector T cells for H-2K or H-2D compatible interactions: implications for H-antigen diversity. Transplantn. Rev. 29, 89-124. Easley C. W. (1965) Combinations of specific color reactions useful in the peptide mapping technique. Biochem. biophys. Acta 107, 36X-388.
Edmundson A. B., Ely K. R., Abola E. E., Schiffer M. & Panagiotopoulos N. (1975) Rotational allomerism and divergent evolution of domains in immunoglobulin light chains. Bi0chemistr.y 14, 3953-3961.
and JOHN J. CEBRA Fairbanks G., Steck T. L. & Wallach D. F. H. (1971) Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistr,, 10, 2606-26
16.
Fellous M., Gunther E., Kemler R., Wiels 3.. Berger R.. Guenot J. L.. Jakob H. & Jacob F. (1978) Association of the H-Y male antigen with /(2-microglobulin on human lymphoid and differentiated mouse teratocarcinoma cell lines. J. exp. Med. 148, 58-70. Gates F. T., Coligan J. E. & Kindt T. J. (1979) Complete amino acid sequence of rabbit fl2-microglobulin. Biochemistry
18, 2267-2272.
Grantham R. (1974)Amino acid difference formula to help explain protein evolution. Science 185, 862-864. Grey H. M., Kubo R. T., Colon S. M., Poulik M. D., Cresswell P., Springer T., Turner M. & Strominger J. L. (1973) The small subunit of HL-A antigens is 82microglobulin. J. up. Med. 138, 1608-1612. Hayashi R., Moore S. & Stein W. H. (1973) Carboxypeptidase from yeast. Large scale preparation and the application to COOH-terminal analysis of peptides and proteins. J. hiol. Chrm. 248, 2296-2302. Helenius A., Morein B., Fries E., Simons K., Robinson P., Schirrmacher V.. Terhorst C. & Strominger J. L. (1978) Human (HLA-A and HLA-B) and murine (H-2K and H-2D) histocompatibility antigens are cell surface receptors for Semliki Forest Virus. Proc. natn. Acad. Sci. U.S.A.
75, 3846-3850.
Isenman D. E., Painter R. H. & Dorrington K. J. (1975) The structure and function of immunoglobulin domains: studies with beta 2-microglobulin on the role of the intrachain disulfide bond. Proc. natn. Acad. Sci. U.S.A. 72, 548-552.
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Note added in proof: The following was inadvertently omitted from the original manuscript: In order to obtain a peptide overlapping peptides T4 through T7, peptide A3 was digested with chymotrypsin. The A3 chymotryptic peptides were fractionated by gel filtration (not shown) which yielded a peptide extending from position 27 (Val) to position 56 (Phe) and was designated A3-Ch2. Automated sequential degradation of A3-Ch2 established the order of tryptic peptides T4 through T7 (Fig. 2).