Turkey β2-microglobulin—I. Isolation, properties and amino acid analysis

Molcwular Immunoloy~. Vol. 19. No. 6. pp. 817427. Punted rn Great Bniain.

0161.5890;82,'060817-I 1803.00,O Pergamon Press Ltd.

1982

TURKEY ~~-MICROGLOBULIN-I. ISOLATION, PROPERTIES AND AMINO ANALYSIS* E. LILLEHOJ,t$$ ~Departm~nt

H. KRUTZSCHll

and M.

D.

ACID

POULIKtT

of Immunology

and Microbiology, Wayne State University School Detroit, MI 48201, U.S.A.; ILaboratory of Immunogenetics, National Institutes of Health, Bethesda, MD 20205, U.S.A.; and IDivision of Immunopathology, William Beaumont Hospital, Royal Oak, Ml 48072, U.S.A.

(first

rrceired

8 June 1981; uccepred

in re~ise~~#r~l

i4 October

of Medicine,

1981)

Abstract-Turkey @,-microglobulin

&m) was purified from pooled serum by successive steps of ultrafiltration, gel filtration chromatography, lectin affinity chromatography, anion exchange chromatography, isoelectric focusing and a second step of gel filtration. Identification of turkey fi2rn was based upon NH~-terminal primary structure analysis. The NH~-terminal primary structure of turkey &rn is: NH,-Lys--Ile-GlumVal-Tyr-IleLys. The purity of the isolated protein was confirmed by two-dimensional polyacrylamide gel electrophoresis, immunodiffusion and immunoelectrophoresis. Physicochemical parameters of turkey Pzm are: mol. wt. 10,500 (observed), 9959 (calculated); /? eiectrophoretic mobility; pl. 4.7, 5.2; E:&. 10.9; absence of terminal D-mannopyranosyl and o-glucopyranosyl residues. Amino acid composition analysis demonstrated similarities between turkey and chicken ,&ms that distinguished them from mammalian fl>rns.

INTRODUCTION

Class I major histocompatibility complex (MHC)** gene products consist of two noncovalently associated polypeptides: a 45,000 mol. wt transmembranal glycoprotein heavy chain and a 12,000 mol. wt peripheral membrane protein, &m. The MHC heavy-chain gene antigenic determinants product expresses recognized by cytotoxic T-lymphocytes during allogeneic (Doherty & Zinkernagel, 1975) and modified self-responses (Shearer, 1974) but the function of Pzm is unknown. However, primary structural studies of human &rn have revealed substantial amino acid sequence homology with both immunoglobulins (Smithies & Poulik, 19720) and HLAs (TrBgrirdh et ul,, 1978) *This work was supported by United States Public Health Service Grant AI-11335 and a grant by the William Beaumont Hospital Research Institute. $Present address: Laboratory of Immunogenetics, Building 8, Room 100, National Institutes of Health, Bethesda, MD 20205, U.S.A. $To whom correspondence should be addressed. **Abbreviations: !??rn. B,-microalobulin; MHC, maior histocompatibility complex_ HLA, lhuman leukocyte antigen; PBS, phosphate-buffered saline; DEAE, diethylaminoethyl; IEF, isoelectric focusing: SDS-PAGE, sodium dodecyl stilfate-polya~rylamide gel electrophoresis; IEP, immunoelectrophoresis: RIA, radioimmunoassay: NRS, normal rabbit serum.

suggesting that this small globular protein may function during immune recognition. Human Pzrn was initially purified from urine of patients with renal tubular dysfunction (Berggbd & Bearn, 1968; Smithies & Poulik, 1972~) and has since been identified in most biological fluids (Talal et al., 1975; Cejka et al., 1976; Vladutiu et al., 1979; Poulik et al., 1979). Analogues of human Bzrn have been purified and studied from a variety of animal species. Complete primary structures are available for human (Cunningham et al., 1973), rabbit (Gates et al., 19791, mouse (Gates et al., 1981) and guinea-pig (Wolfe & Cebra, 1980) &ms as well as NH~-terminal residue sequences of dog (Smithies & Poulik, 1972b), rat (Poulik et al., 1979) and cow (Groves & Greenburg, 1977) /&ms. These data reveal substantial interspecies homology. Most of the differences that are observed can be accounted for by single DNA base pair substitution (Gates et al., 1979, 1981) although nonmammalian Pzms have not been investigated in this respect. Previous investigations have demonstrated a small mol. wt protein noncovalently bound to chicken B-locus MHC antigens (Ziegler & Pink, 1975). Winkler & Sanders (1977) have described the isolation of a chicken serum protein

E. LILLEHOJ.

818

H. KKUTZSCH

with characteristics of fizm. The present study was undertaken to purify turkey fi2rn for comparison with chicken and mammalian &ms. MATERIALS

AND

and M. D. POULIK

o~~albumin ~45,~), chymotrypsinogen A (25,000), ribonuclease A (13,700) and human Pam (previously purified in this laboratory, 11,800).

METHODS

Lentif &tin a~n~ty chromatography Positive pressure ultrafiltration was performed using Amicon XM-50 hollow fiber filters (50,000 mol. wt retention, Amicon Corp., Lexington, Massachusetts). Pooled turkey serum (Pel Freez Biologicals, Rogers, Arkansas; Bel-Mar Turkey Farm, Zeeland, Michigan) was centrifuged at 8000 g for 30 min at 4”C, 2-l. aliquots diluted 1: 2 (v/v) with deionized water and subjected to ultrafiltration at 22’C until the retenate was reduced to 2 1. This retenate was again diluted 1:2, the procedure repeated 2 or 3 times and the final filtrate comprising 4-8 1. was concentrated by lyophilization (Virtis Co.. Gardiner, New York). Gel jiltration

chromatography

This was performed using Biogel P-100 (100-200 mesh, Bio-Rad Laboratories, Richmond? California) or Sephadex@ G-50 (superfine, Pharmacia Fine Chemicals Inc., Uppsala, Sweden). Elution buffer was either 0.1 M NH,HCO,, 0.1% NaNs, pH 8.5, or 0.01 M Tris-HCl, 0.5 M NaCl, 0.1% NaN,, pH 7.4. The XM-50 filtrate of turkey serum was routinely gel filtered at 22°C on Biogel P-100 columns (5 x 87-91 cm) at a flow rate of 20-25 ml/hr and fractions of 6.7-8.3 ml collected (LKB 7000 Ultrorac@ fraction collector, LKB-Produkter AB, Bromma, Sweden). Gel filtration chromatography employing Sephadex@ G-50 was accomplished on 1.8 x 93 cm columns at a flow rate of 3.0 ml/hr collecting 3.0-ml fractions. Protein elution was monitored by transmittance at 280nm (%l;sO, Uvicord II, LKB-Produkter AB) or absorbance at 280 nm (A2s0, PM6 spectralphotometer, Carl Zeiss, Oberkochen, West Germany). Individual fractions were pooled, dialysed against deionized water (Spectrapor@ 3 dialysis tubing, 600&8000 mol. wt retention, Spectrum Medical Industries Inc., Los Angeles, California) at 4°C and lyophilized. Mol. wt determinations were made according to the method of Andrews (1964) using the following mol. wt standards (Pharmacia): blue dextran (2,000,000), bovine serum albumin (66,200)

Low mol. wt proteins from Biogel P-100 columns were fractionated on the lectin from Lens culinaris covalently coupled to Sepharose@ 4B (Pharmacia) according to the procedure of Hayman & Crumpton (1972). Affinity chromatography was performed using PBS (0.01 M sodium phosphate, 0.15 M NaCI), pH 7.2. Following elution of unbound proteins, bound. proteins were desorbed with PBS, pH 7.2, containing 0.2 M ff-methylmannoside (Sigma Chemical Co., St. Louis, Missouri). Anion exchange

chromatography

This was performed using DEAE-cellulose ~hatman Inc., Reeve Angle, California) according to the manufacturer’s instructions with the following modifications. Before application. the protein sample was dialysed at 4°C against starting elution buffer (0.01 M TrisHCI, pH 8.0) until the conductivity of the sample and buffer were identical (CDM 3 conductivity meter, Radiometer, Copenhagen, Denmark). Insoluble material was removed by centrifugation (10,000 g, 30 min, 22°C) and the sample applied to a 0.9 x 25.0 cm column preequilibrated with starting buffer. After elution of unbound proteins, a linear gradient from 0 to 0.2 M NaCl was established in 0.01 M Tris-HCl, pH 8.0 (GM-l gradient mixer, Pharmacia). The flow rate was maintained at 9.3 ml/hr collecting fractions of 4.7 ml at 22°C. Conductivity of selected fractions was measured to determine the NaCl gradient. Protein elution was monitored by %T2s0. Individual fractions were pooled, dialysed against deionized water and lyophilized. IEF

Preparative IEF (8101 column, LKB-Produkter AB) was performed according to Vesterberg (1971) using pH 4-6 and 2-10 ampholytes (Brinkman Instruments, Westburg, New York) at 5% (w/v) final concentration. Electrophoresis proceeded for 60-72 hr at 9°C maintaining a constant power of 2.8-3.0 W (model 3371E D.C. power supply, LKB-Produkter AB). The

Turkey

P2-Microglobulin--I

current at the end of the experiment was less than 0.2 mA. Protein elution was monitored by A280‘ Individual fractions were pooled, dialysed and freeze-dried as earlier. Protein determination The method of Lowry et al. (1951) was used with bovine serum albumin (crystallized and lyophilized, 76C-8130, Sigma) as standard. The concentration of purified proteins was determined from Azso measurements based on published E:& values (Edelhoch, 1967).

819

injected at four intramuscular dorsal sites. All rabbits were boosted twice at 2-week intervals, bled to assess antiserum activity and boosted again if necessary. Serum was separated by centrifugation at 1200~ for 20min and stored at -20°C. Double immunodiffusion was performed as described by Ouchterlony & Nilsson (1978) with 1% (w/v) agarose (Marine Colloids, Rockland, Maine) in PBS at pH 7.2. IEP was accomplished by the method of Scheidegger (1955) with 1% Oxoid Ionagar No. 2 (Colab Laboratories, Chicago Heights, Illinois) in barbital buffer (p = 0.05) containing 0.05% NaN, at pH 8.6.

Iodogen@ catalysed protein ~odinution Fifty microliters (5-5Opg) of purified ,!&rn in PBS, pH 7.2, was labeled with 1.0 mCi of carrier free Na’ 251 (13-l 7 mCi/mg, AmershamSearle, Arlington Heights, Illinois) using 1,3,4,6tetrachloro-3~,4~-diphenylglycouril (Iodogen@, Pierce Chemical Co., Rockford, Illinois) according to the method of Fraker & Speck (1978) slightly modified in this laboratory. Bound and free “‘1 were separated on 0.2 x 6.0cm columns of Biogel P-2 (Bio-Rad) overlaid with l.Ocm of Sephadex* G-25 (Pharmacia) and precoated with 25 mg of ovalbumin (Sigma) in PBS, Both precipitation with 7% trichloroacetic acid and paper chromatography in 857; methanol routinely indicated 63-8576 proteinbound lzsI in the unfractionated sample and 83-977: bound lzsI in the major gel filtration fraction. The specific activity of radioiodinated pzrn was 0.33-0.70 mCi/mg.

Amino acid composition analysis Selected proteins were analysed for amino acid content in collaboration with Dr Dan Walz, Department of Physiology, Wayne State University, Detroit, Michigan, and Dr Jan Cejka, Department of Immunochemistry, Children’s Hospital of Michigan, Detroit, Michigan. Protein samples were hydrolysed in 6 N HCl for 24 or 72 hr in uacrto and analysed on a Beckman 119CL amino acid analyzer (Beckman Instruments Inc., Wakefield, Massachusetts) using a one-column procedure (column dimensions, 6 x 210mm). Amino acids were eluted with a three-buffer system using lithium citrate (0.2 N, pH 2.83; 0.2 N, pH 3.70; l.ON, pH 3.75) and a two-temperature procedure (40, 65°C). Tryptophan was determined spectrophotometrically (Edelhoch, 1967). Amino-term&a/ amino acid determination

The procedure of O’Farrell et al. (1977) was used for nonequilibrium pH gel electrophoresis in 2.5 x 130 mm cylindrical gels and the procedure of Jones (1977) utilized for discontinuous SDS-PAGE on 11.7 x 13.5 cm 12.5% (w/v) acrylamide slab gels. Following electrophoresis, the gels were dried and exposed to Kodak Ortho G X-ray film (Eastman Kodak Co., Rochester, New York) at -80°C for 3 days. immunological methods Rabbits were immunized with antigen solutions in PBS, pH 7.2, at 250 pg/ml. One milliliter was emulsified in 1.0 ml Freund’s complete adjuvant (Difco, Detroit, Michigan) and

The dansylation procedure described by Gros & Labouesse (1969) was used. Dansylated amino acids were resolved by two-dimensional thin-layer chromatography on 5 x 5 cm polyamide sheets as described by Zimmer et al. (1976) using water:formic acid (50: 1.5, v/v) as the first-dimension solvent and benzene: acetic acid (9: 1, v/v) for the second dimension. Edman amino-terminal analysis

amino acid sequence

This was performed according to the published methodology (Gates et al., 1979) using a Beckman 890C sequencer and DMAA program (No. 102974). Phenylthiazolinone derivatives were converted to phenylthiohydantoin amino acids by heating at 80°C for 5 min in

820

E. LILLEHOJ,

H. KRUTZSCH

1 N HCl and under Nz, and identified by highpressure liquid chromatography at 254 nm on a Waters Associates (model 402) chromatograph equipped with a DuPont Zorbax ODS column (4 x 250 mm) using a modification (Gates er ul., 1979) of the method of Zimmerman et al. (1977).

RESULTS

Purification

of turkell flzrn

The isolation of turkey fi2rn utilized pooled serum that was subjected to sequential steps of Amicon XM-50 ultrafiltration, Biogel P-100 gel filtration chromatography, lentil lectin affinity Whatman DE-52 ion chromatography, and chromatography, IEF exchange Sephadex@ G-50 gel filtration chromatography. Two assumptions were made with the first two fractionation steps. First. the mol. wt of turkey fizrn was assumed to be similar to that of known mammalian b2ms, approximately 12,000. Turkey Bzrn was also assumed not to be a glycoprotein. All p2rns that have been isolated and characterized share these features. The 50,000 mol. wt filtrate obtained by ultrafiltration was concentrated by lyophilization and gel filtered in the presence of ‘251-human P2rn (Fig. 1). Pools V and VI coeluting with human Pzrn were combined and fractionated on lentil lec-

10

c

I

and M. D. POULIK

tin-Sepharose@ 4B to remove glycoproteins. Greater than 99% of the applied protein appeared in the unbound fraction from this column (data not shown). Although this step did not substantially improve the purification of turkey /$rn, it did ensure that the final product lacked terminal o-mannopyranosyl and o-glucopyranosyl residues. However, we cannot exclude the presence of other carbohydrate moieties. The unbound pool obtained from lectin affinity chromatography was subsequently fractionated on DEAE-cellulose (Fig. 2) and several pooled fractions from this column were submitted for NH,-terminal amino acid sequence analysis. Identification of turkey P2rn was based on comparison of these data with known primary structural information available for various fl*rns. Anion exchange chromatography pool B2071 VI was shown to with NH,-terminal possess a polypeptide lysine and a sequence homologous to mammalian &ms (see later). In addition, three other NH,-terminal residues were detected in this pool (val, gly, glu) indicating the presence of impurities. Accordingly, preparative IEF and Sephadex@ G-50 gel filtration were undertaken to remove these contaminants. Fig. 3 shows the elution profile of DEAE-cellulose pool B2071 VI resolved by IEF over a pH gradient of 367.5. Fractions from this step were pooled, dialysed against deionized water and analysed for NH,-terminal amino acid residues using

,

I

VO

45.0kd I

ELUTION

25.0kd I

VOLUME

I

ll.Bkd I

(ml)

Fig. I. Gel filtration chromatography elution profile (B2061) of the Amicon XM-50 filtrate of pooled turkey serum containing ‘251-lab~led human Bzrn on Biogel P-100 (5 x 87cm). Elution buffer’0.1 M NH,HC03, O.lp!, NaN3, pH 8.5. Fractions of 4.5 ml collected at a flow rate of 20 ml/hr. Mol. wt markers mdicated in kilodaltons (kd).

821

Turkey /32-Microglobulin-

I-

82071 / / / 0

t_

ELUTION

VOLUME (ml)

Fig. 2. Anion exchange chromatography elurion profile (B2071) of the unbound fraction from lentil lectin affinity chromatography on DEAE-ceIl~~iose (0.9 x 25 cm). Equilibrating buffer 0.01 M Tris-HC1, pH 8.0, and the limiting buffer 0.01 M Tris--HCl containing 0.2 M NaCI. Fractions of 4.7 ml collected at a flow rate of 9.3 mljhr.

dansyl chloride, NH2-Terminal lysine, characteristic of turkey /&rn, was identified in pool II (Fig. 3). The other fractions possessed different amino acids as the major labeled residue with the exception of pool IV in which an identification could not be made possibly due to a blocked NH,-terminal amino group. However, we do not feel that turkey Pzm is present in pool IV because: (i) the isoelectric point of the major absorbance peak in this pool is less than

030 =

that for any known mammalian /Izm, and (ii) further analysis of pool II confirmed the presence of /12rn (see later). Two-dimensional gel electrophoretic analysis of pool IEF 071880 II revealed a minor contaminant with a similar isoelectric point but greater mol. wt than turkey &m (data not shown). In an attempt to remove this impurity, the f12rn preparation was applied to a column of Sephadex@ G-50 and eluted as described in

-T----T--.

‘-7

K

IEF 071880

0 25

020

$1

015

-

010

-

005

-

20

40

ELUTION

60

VOLUME

80

100

(ml)

Fig. 3. Isoelectric focusing elution protile (IEF 071880) of DEAE-cellulose pool B2071 VI obtained after 67 hr at 9’C using ampholytes pH 4-6 (I.t”,;) and pH 2-10 (0.80,,,).Fractions of 2.0 ml collected for protein (Ala,,; 0-O) and pH (i-+) measurements. NH~-Terminal amino acid determined for each pool is indicated.

E. LILLEHOJ,

822

H. KRUTZSCH and M. D. POULIK

yi”

13.i”d

1

,ti” 82114 i

150

100

50

ELUTION VOLUME (ml)

Fig. 4. Gel filtration chromatography elution profile (82114) of isoelectric focusing fraction 071880 II on Sephadew@ G-SO (1.6 x 93 cm). Elution buffer 0.01 M Tris-HCI, 0.5 M NaCI, 0.17; NaN,, pH 7.4. Fractions of 3.0 ml collected at a flow rate of 3.0 ml/hr. Mol. wt markers indicated in kilodaltons (kd).

10,500 and this was confirmed by SDS-PAGE (data not shown). The small recovery of turkey &m mandated a second larger-scale isolation scheme which was accomplished after developing a competitive binding RIA (Lillehoj & Poulik, 1982) used to monitor purification steps identical to those

Fig. 4. Fractions eluting between 94 and 130 ml were combined (pool II), dialysed and lyophilized, resulting in approximately 200 ,ug of highly purified turkey Pzm. Amino acid composition analysis confirmed the presence of flzrn (see later). The apparent mol. wt determined by the method of Andrews (1964) was

Table I. Summary of turkey /&rn purification from turkey serum Specific activity*

Step

Fraction

A,,0

Amicon XM-50 ultra~ltration

Crude Filtrate Retenate

28.5 1.44 63.4

24.8 11.3 74.1

0.87 7.85 1.17

CO’

107 1.69 2.16

126 0.35 0.19

1.18 0.21 0.09

III IV V VI

2.76 0.93 0.09 0.088

0.20 0.10 0.61 5.93

CO

1.61 0.043 0.047 0.049 0.086 0.184 0.109 0.091

I II Biogel P-100

Whatman DE-52

Sephadex@ G-50

I II III IV V VI VII F”

2.42 0.11

606 3.29 11.8 2.42 16.1 222 22.3 26.0

2850 125

“Determined by RIA (Lillehoj & Poulik, 1982). %pecific activity (SA) = &ml /&m + Azso. ‘Purification = greatest SA fraction c CO SA. ‘Yield = total fizm recovered + total Pzm applied. “CO = column original material.

0.07 0.11 6.78 67.4 376 16.5 251 49.4 187 1207 214 286

1178 1190

Purification (fold)

Yield (?“Jd

9.0

90.1

_

514

52.1

1644

36.7

1696

6.4

823

Turkey [jz-Microglobulin- I described earlier. A summary of the second isolation scheme is presented in Table 1. Although the first and second isolation schemes utilized common purification steps, individual pools between the two protocols do not coincide. Thus, while the N-terminal amino acid sequence of turkey /12rn was derived from pool B2071 VI, it can be seen in Table 1 that DE-52 pool V from the second scheme is richer in /3,m. Furthermore, although Sephadex@ G-50 chromatography illustrated in Fig. 4 shows two pools, only one containing B2rn was prepared in the second scheme.

Analysis of protein purity Two-dimensional gel electrophoresis of purified i2’I-labeled turkey jIzrn revealed two components of identical mol. wt and distinct isoelectic points (pZ 4.7, 5.2; Fig. 5). Charge of chicken (Winkler & polymorphisms Sanders, 1977) rat (Liigdberg et ul., 1979) mouse (Michaelson et al., 1980) guinea-pig (Wolfe & Cebra, 1980) and human (Hall et cl/., 1977) p2ms are well documented, but we cannot exclude the presence of a contaminant particularly since pool B2114 II is not homogeneous in size. The purity of turkey film was further investigated by immunodiffusion (Fig. 6A) and IEP (Fig. 6B). Rabbit antiserum DP412 prepared against the original DEAEcellulose column fraction containing b2m (pool

B2071 VI) precipitated three distinct components in this crude fraction. However, only a single precipitation line with p mobility was detected against purified turkey /Izrn (pool B2114 II). NRS did not react with either the crude or purified antigen preparations. From these results we conclude that isolated turkey fi2rn is free of detectable impurities. Amino acid analysis The amino acid composition of turkey /Izrn (pool B2114 II) is compared with those of chicken and mammalian P2ms in Table 2. Based upon these results, the extinction coefficient (_E:&) of turkey pzrn is 10.9 (Edelhoch, 1967) and the calculated mol. wt is 9959. Table 3 lists the NH,-terminal amino acid sequences of DEAE-cellulose pooled fraction B2071 VI for comparison with human and dog P2ms. Pool 2071 VI is similar to the mammalian polypeptides although a gap in the first five residues of the mammalian proteins is required to align them for maximum homology.

DISCUSSION Initial attempts to purify turkey /12rn were founded on several assumptions. Although urine has consistently served as the primary starting material for purification of Pzm from

p! Pj

!I

-

Fig. 5. Two-dimensional

gel electrophoresis

of ‘251-labeled

turkey

21,500

Btm (pool B2114 II)

824

E. LILLEHOJ,

H. KRUTZSCH

and M. D. POULIK

Fig. 6. Ouchterlony double immunodiffusion (A) and immunoelectrophoresis (B) of crude (c) and purified (p) turkey bzm. Concentrations are indicated for each preparation. Rabbit antiserum DP 412 was prepared against the crude f12rn preparation (pool B2071 VI) and used undiluted. NRS, normal rabbit serum.

mammals with renal tubular disease, avian urine combined with solid waste is unsuitable. Thus, serum was chosen as the source of turkey /&rn and it was assumed that mammalian and avian serum flzrn content were similar. The extent of fl,-microglobulinemia in humans has been determined to be 1.0-2.5 pg/ml (Evrin & Wibell, 1972) therefore necessitating large quantities of this fluid in the present study. After development of a competitive binding RIA specific for turkey Pzrn, a much greater discrimination in pooling fractions containing p2rn was made during the second purification scheme. Therefore, while six separate fractionation steps were required for final purification of turkey p2rn in the first scheme, only four were needed for the second protocol. In contrast to molecular size and absence of

glycosyl residues, the charge characteristics of mammalian f12rns are heterogeneous. For instance, rat /3,m migrates as a y-globulin which is reflected in a higher isoelectric point Cpl7.4; Logdberg et al. (1979)] compared to human p2rn [PI 5.7; Hall et al. (1977)]. Both proteins additionally possess a minor, more acidic form (rat f12rn, pZ6.8; human f12rn, ~15.3). Other &ms have also been shown to possess two electrophoretic components (Cigen et al., 1978; Michaelson et al., 1980; Winkler & Sanders, 1977). Two-dimensional gel electrophoresis of purified turkey b2rn (pool B2114 II) revealed two components of identical mol. wt but different charge (pZ 4.7, 5.2). The mechanism(s) responsible for charge variations of different /&rn preparations is unknown but several have been proposed. Evidence for mur-

Turkey

Table 2. Comparison

of amino

acid compositions

Human”

Rabbitb

Mouse’

Asx Thr Ser Glx Pro GIY Ala Val ) Cys Met Ile Leu Tyr Phe Lys His

12 S 10 11

8 4

15 4 6 12 6 3 2 10 2 1 3 7 5 5 8 4

10 8 6 11 9 2 3 5 2 5 7 3 5 4 9 5

Trp Arg Total

52 100

42 99

: 99

; 2 7 2 : I :

B,-Microglobulin-

Guinea-pigd 13 3 9 9 7 3 4 9 2

1 5 7 4 5 8 5 3 2 99

825 of /&rns from various

animal

Rat’

Cow’

Dogs

Chicken”

10 8 6 13 9 2 2 6 2 2 7 6 4 S 9 4

11 2 8 12 9 3 1 5 2 0 6 8 6 4 9 4

: 100

: 97

12 6 7 14 8 6 4 7 2 1 3 7 3 6 8 4 3 2 103

14 6 7 14 8 7 7 8 3 2 3 7 3 4 5 1 4 2 105

species Turkey’ 9 5j 13’ 13 4 13 7 4k 21 0 3’ 6

1 3 6 1 3 2’ 95

“From amino acid sequence (Cunningham et al., 1973). *From amino acid sequence (Gates et al., 1979). ‘From amino acid sequence (Gates er al., 1981). “From amino acid sequence (Wolfe & Cebra, 1980). ‘Logdberg r)t ai. (1979). /Groves & Greenberg (1977). gPoulik (1973). hWinkler & Sanders (1977). ‘One 24-hr and one 72-hr hydolysis of pool B2114 II. ‘Extrapolated to O-hr hydrolysis value. ‘Seventy-two-hour hydrolysis value only. ‘Determined spe~trophoton~etrically (Edelhoch. 1967).

e /&rn strain-related primary structure polyorphism has been presented by Gates et al. 981). Complete amino acid sequence data of rinea-pig pzrn has also revealed polymornsm (Wolfe & Cebra, 1980). Alternatively, large variations may follow post-translational :amidation and/or proteolysis. This investiga)n did not attempt to isolate and identify the 70 apparent charge forms of turkey Pam though such studies are clearly warranted to

Table

Human

3. NH,-Terminal

PZmb

Dog P*rn B2071 VI“

completely exclude the possibility of copurification of different polypeptides. The amino acid composition of turkey /I&m shows certain similarities and differences with chicken and mammalian pzms. The avian proteins are distinguished from mammalian &ms in that both possess relatively decreased amounts of isoleucine, tyrosine, lysine and histidine as well as a larger content of alanine. Glycine is also relatively abundant in the avian

amino acid sequence of turkey with human and dog Pzms”

j&m compared

1 10 I-Q-R-T-P-K-I-Q-VY-S-R10 :mQ-H-PPP-K-I-QVY-S-R5 1 K-I-E-V-Y-I-K

“Single-letter designations of amino acids are used (IUPAC-IUB Commission on Biochemical Nomenclature, 1968); A, ala; R, arg; D, asp; C, cys; Q. gln: E. glu: G, gly; H, his; I, ile; L. leu; K. lys; M, met; F, phe; N, asn: P. pro: S, ser; T, thr: W, trp: T, tyr: V, val. “Cunningham cr cd. (1973). ‘Smithies and Poulik (1972h). ‘Aligned for maximum homology with mammalian blms.

826

E. LILLEHOJ.

H. KRUTZSCH

&rns although the value obtained in this study may be artifactually large. Important differences between chicken and turkey Pzms are also apparent. Chicken flzrn possesses three cysteine residues compared to two for turkey and mammalian b2ms. All ljzms most likely possess a single disulfide loop similar to the immunoglobulin domain and this has been confirmed in human (Cunningham et al., 1973), rabbit (Gates et at., 1979), mouse (Gates rt al., 1981), guinea-pig (Wolfe & Cebra, 1980) and rat (Liigdberg er al., 1979) films. The presence of a free sulfhydryl group in chicken fi2rn suggests the possibility of disulfide linkage to the histocompatibiIity heavy chain which would further substantiate the immunoglobulin-like structure of classs 1 MHC antigens that has been proposed to exist at both the primary (Trsg%rdh ef al., 1978) and tertiary (Strominger et nl., 1974) structural levels. Furthermore, turkey fi2rn appears more simiIar to the human protein than chicken or other mammalian /3,ms on the basis of the number of certain residues, particularly proiine and serine. The apparent mol. wt of turkey j&m was 10,500 after gel permeation chromatography and SDS-PAGE. In contrast, the calculated mol. wt is 9959. A molecular size discrepancy of approximately 1000 also exists between turkey and mammalian /&rns which may in part be accounted for by primary structural data. A deletion encompassing residues l-5 of mammalian &ms is required to align turkey and mammalian fizms for maximum homology. This suggests that native turkey ,&,m may be composed of 94 total amino acids possibly due to avian-related mechanisms of signal peptidase activity (Lingappa et at., 1979) and/or nucleic acid processing (Moore et al., 1981). Interestingly, partial primary structural data of mammalian azms indicates greater interspecies variability between residues 1-4 than 5-12, suggesting that the first few NH,-terminal residues may not substantially contribute to protein function. Alternatively, turkey /&rn may be highly susceptible to exopeptidase attack although we could find no evidence for proteolytic degradation of human j&m by turkey serum (data not shown), or larger mol. wt forms of [Izrn in turkey serum (Lillehoj & PouLik, 1982).

In light of several ~hysicochemical dissimilarities between turkey and mammalian j!&rns described in this study, it was of interest to extend this comparison by other criteria. The

and M. D. POULIK

second paper in this series examines the physiological and immunological parameters of turkey f12rn by RIA and confirms avian-specific characteristics of this protein. Ackno~iniyetnmrs--The authors thank Drs D. Walz and J. Cejka for amino acid composition analysis, and Miss D. M. Casper for her excellent secretarial assistance.

REFERENCES Andrews P. (1964) Estimation of molecular weights of proteins by Sephadex gel-filtration. Biochem. J. 91, 222-223. Berggird I. & Bearn A. (f968) Isolation and properties of IOW molecular weight fiz-globulin occurring in human biological fluids. J. hiol. Chem. 243, 4095-4103. Cejka J., Van Nieuwkoop J., Mood D., Kithier K. & Rad] J. (1976) &-Microglobulin in human colostrum and milk: effect of breast feeding and physiochemical characterization. CIinica chim. Acta 67, 71-78. Cigtn R., Ziffer J., Bergg%rd B., Cunningham B. & BerggHrd I. (1978) Guinea pig P,-microglobulin. Purification, properties and partial structure. Biochemistry 17, 947-955. Cunningham B., Wang J., Berggard I. & Peterson P. (1973) A complete amino acid sequence of ~~-microglobulin. ~iffc~e~~sf~.~ It, 481 I-4822. Doherty P. & Zinkernagel R. (1975) H-2 compatibility is required for T cell-mediated lysis of target cells infected with lymphocytic choriomeningitis virus. J. exp. Med. 141, 502-507. Edelhoch H. (1967) Spectroscopic determinatjon of tryptophan and tyrosine in proteins. Biochemistry 4, 1948-1954. Evrin P. & Wibel] L. (1972) The serum levels and urinary excretion of fi2-microglobulin in apparently healthy subjects. Stand. J. clin. Lab. Invest. 29, 69-74. Fraker F. & Speck J. (1978) Protein and cell membrane iodinations with a sparingly soluble chloroamide, Biochem. 1,3,4,6-tetrachloro-3c,6&diph&yIglycoluril. biophvs. Res. Commun. 80, 849-857. Gates e., Coligan J. & Kindt T. (1979) Complete amino acid sequence of rabbit fi,-microglobulin. Biochemistry 18, 2267-2272. Gates F., Coligan J. & Kindt T. (1981) Complete amino acid sequence of murine &-microglobulin: structural evidence for strain-related polymorphism. Proc. natn. Acad. Sci. U.S.A. 78, 554-558. Gros C. & Labouesse B. (1979) Study of the dansylation reactions of amino acids, peptides and proteins. Eur. J. Bioc~zem. 7, 463-470. Groves M. & Greenburg R. (1977) Bovine homologue of p,-microglobulin isolated from milk. Biochem. biophys. Res. Commun. 77, 320-321. Hail P., Ricanati E. & Vacca C. (1977) Characterization of human &-microglobulin by isoelectric focusing. Cfinica chim. Acta 77, 3742. Hayman M. & Crumpton M. (1972) Isolation of glycoproteins from pig lymphocyte plasma membranes using Lens cufinaris phytohemagglutinin. Biochem. biophys. Res. Commun. 47, 923-930. Jones P. (1977) Analysis of H-2 and Ia molecules by twodimensional gel electrophoresis. f. exp. Med. 146, 1261-1219. Lillehoj E. & Poulik M. (1982) Turkey bz-microglobulinII. Physiological and immunological parameters investigated by radioimmunoassay. Molrc. Immuti. 19, 829.-837.

Turkey

&Microglobulin--I

Lingappa V., Cunningham B., Jazwinski M., Hoop T., Blobel G. & Edelman C. (1979) Cell-free synthesis and segregation of /&-microglobulin. Proc. narn. Acod. Sri. U.S.A. 76, 3651-3655. Liigdberg L., ijstergren P.-O. & Berg&d I. (1979) Rat fl,-microglobulin. isolation, properties and relation to P,-microglobulins from other species. Moles. Immun. 16, 577-587. Lowry 0. H., Rosebrough N. J., Farr A. L. & Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Michaelson J., Rothenberg E. & Boyse E. (1980) Genetic polymorphism of ~~-microglobulin. J~m~nogenefic~ 11, 93-95. Moore K., Rogers J., Hunkapillar T., Early P., Nottenberg C., Weissman I., Bazin H., Wall R. & Hood L. (1981) Expression of IgD may use both DNA rearrangement and RNA splitting mechanisms, Proc. natn. Acad. Sci. U.S.A. 78, 18OC-1804. O’Farrell P., Goodman H. & O’Farrell P. (1977) High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell 12, 1133-l 141. Ouchterlony 0. & Nilsson L. (1978) Immunoditlusion and immunoelectrophoresis. In Handbook of Experimental Immunology (Edited by Weir D.), p. 19.10. Blackwells, Oxford. Poulik M. (1973) Presence of ~~-micro~lobulin on B- and T-celis and Iymphocytotoxicity of ant]-~~-microglobulin sera. Immun. Commun. 2, 403-414. Poulik M., Gold P. & Shuster J. (1979) Methods for and clinical applications of p,-microglobulin. Crir. Rev. c/in. Lab. Sci. 10, 225-245. Poulik M. D. & Smithies 0. (1979) Partial amino acid sequences of rabbit and rat ~~-microglobulins. Molec. rF~rn~~. 16, 731-734. Scheidegger J. (1955) Une micro-methode de I’immunoelectrophoresis. INI. Arch.7 AIIergy appl. Immun. 7, 103-110. Shearer G. (1974) Cell mediated cytotoxicity to trinitrophenyl-modified syngeneic lymphocytes. Eur. 1. Immun. 4, 527-533.

827

Smithies 0. & Poulik M. (1972a) Initiation of protein synthesis at an unusual position in an immunoglobulin gene. Science 175, 187-189. Smithies 0. & Poulik M. (1972b) Dog homologue of ~*-microglobulin. Proc. mtn. Acad. Sci. U.S.A. 69, 29142917. Strominger J., Cresswell P., Grey H., Humphreys R., Mann D., McClune J., Parham P., Robb R., Sanderson A., Springer T., Terhorst C. & Turner M. (1974) The immunoglobulin-like structure of human bistocompatibility antigens. ~ra~sp~a~tn Reu. 21, 126-143. Talal N., Grey H., Zjaijler N., Michalski J. & Daniels T. (1975) Elevated salivary and synovial fluid Pz-microglobulin in Sjogren’s syndrome and rheumatoid arthritis. Science 187, 1196--l 198. Tr6gBrdh L., Wiman K., Rask L. & Peterson P. (1978) Amino acid sequence homology between HLA-A,B,C antigens, /?,-microglobulin and immunoglobulins. Stand. J. immun. 8, 563.-568. Vesterberg 0. (1971) Isoelectric focusing of proteins. i2lerh. Enzym. 22, 389-412. Vladutiu A., Adler R. & Brason F. (1979) Diagnostic value of biochemical analysis of pleural effusions. Carcinoem: bryonic antigen and P,-microglobulin. Am. J. c/in. Parh. 71, 210-214. Winkler M. & Sanders B. (1977) Chemical and immunologic characterisation of a ~~-microglobulin-like protein isolated from chicken sera. ~mrn~~o~hernistr~~ 14, 615-619. Wolfe P. B. & Cebra J. J. (1980) The primary structure of guinea pig p2-microglobulin. Mo/ec. Immun. 17, 1493-1505. Ziegler A. & Pink R. (1975) Characterization of major histocompatibility (B) antigens of the chicken. TrarwlantaEion 26, 523-527. _ :Zimmer H., Neuhoff V. & Schulze E. (1976) Low-fluorescence polyamide sheets for thin-layer chromatography. J. Chromat. 124. 120-122. Zimmerman C., Appella E. & Pisano J. (1977) Rapid analysis of amino acid phenylthiohydantoins by high-pressure liquid chromatography. Anaiyt. Biochem. 77, 569-573.