Biochimica et Biophysica Acta, 317 (1973) 447-461
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 36492 CYANOGEN BROMIDE S P L I T T I N G OF HUMAN IMMUNOGLOBULIN M
JIl~t ZIK~_N* AND
J.
CLAUDE BENNETT
Division of Clinical Immunology and Rheumatology, Departments of Medicine and Microbiology, University of Alabama in Birmingham, University Station, Birmingham, AIa. 35294 (U.S.A.)
(Received March 5th, 1973)
SUMMARY Human Waldenstr6m IgM (K) (Dau), its polypeptide chains and its F(c)5 # and Fab # fragments were split by cyanogen bromide (CNBr). The fragments formed by CNBr were fractionated by gel filtration, ion exchange chromatography and paper electrophoresis. They were characterized in terms of polyacrylamide disc electrophoresis, peptide maps, amino acid composition, end group determinations and limited primary structure determination. Two CNBr fragments were formed from the K chain, consistent with the presence of one methionine residue. Five fragments were isolated from the partially S-sulfonated F chain. Three additional fragments were released after destruction of all disulfide bonds. The present data are unclear as to whether there are eight or nine CNBr fragments released. The comparison of CNBr pieces from the IgM, # chain, F(c) 5/z and Fab # affords a tentative arrangement of their order, as well as the relative location of the disulfide bonds within the molecule.
INTRODUCTION Determination of the primary structure of IgM requires splitting of this large molecule into smaller, more workable fragments. With this molecule, as with other immunoglobulin classes, the first step has been the isolation of fragments following disruption of either interchain disulfide bonds or cleavage of labile peptide bonds in the hinge region. These manipulations lead to the production of H(F ) and L(K) chains, and F(c)5 # and Fab # fragments, respectively1,2. These fragments, as well as whole IgM, may be broken down further by cyanogen bromide (CNBr) which specifically splits peptide bonds on the carboxyl side of methionine, converting it into homoserine lactone 3. This report describes the isolation and characterization of these fragments produced by CNBr cleavage. * Present address: The Institute of Microbiology, Czechoslovak Academy of Sciences, Prague, Czechoslovakia.
448
j. zlKAN, j. c. BENNETT
MATERIALS AND METHODS
Preparation of IgM and its polyp@tide chains and fragments H u m a n Waldenstr6m IgM (K) (Dau) was prepared as described by BennetO. The component/~ and K chains were separated in 5 M guanidine. HC1 after splitting of disulfide bonds by partial oxidative sulfitolysisl, a. The F(c)~ # and Fab # fragments were separated on Sephadex G-2oo in I°:,/o NH4HCO3, p H 8, after hot trypsin digestion 2 as modified from Plaut and Tomasi 6.
CNBr cleavage CNBr cleavage of IgM and its subunits was performed by a modification of the method of Waxdal et al. 7. The protein sample (2o 5o mg) was dissolved in I nfl of 7o% formic acid and a 2-fold (w/w) amount of CNBr was added. Nonspecific splitting was minimized by carrying out the reaction in an ice bath. Analysis of methionine and homoserine residues at different stages of CNBr cleavage of the # chain revealed that the reaction was finished at 4 °C in 8 h, when 98% of the methionine residues were modified to homoserine lactone.
Analytical methods Fractions were assayed for homogeneity by disc electrophoresis in sodium dodecyl sulfate 8. Molecular weights were determined on gels according to the technique of Weber and Osborn 9. Disc electrophoresis was performed in both alkaline 1° and acid urea n. Paper high voltage eleetrophoresis was performed in pyridine acetate buffer (pH 3.6) as described by BennetO 2. Immunoelectrophoresis was performed according to the method of Scheiddeger, as described by Grabar ~3, using 1.5% agarose in 0.05 ionic strength sodium barbital buffer (pH 8.6) as the gel medium. Precipitating goat anti-K and anti-# antisera were obtained from Melpar Inc., Falls Church, Va., U.S.A. The gel filtrations were performed with upward flowing Sephadex columns. Molecular weight determinations were done according to Andrews 14 using standardized columns. High speed short column sedimentation equilibrium experiments were performed and weight average molecular weights were calculated according to Yphantis 15. In all cases the partial specific volume was assumed to be 0.74. Samples for amino acid analysis were hydrolyzed in constant boiling HC1 at IiO °C in vacuum sealed tubes for 2o and 48 h. After evaporation of the HC1, the sample was analyzed by standard procedures on a Beckman I2oC amino acid analyzer. Every analysis was done at least twice and average values were calculated. The losses of threonine and serine during hydrolysis were estimated by extrapolation to zero time. The determination of half cystine residues in the form of cysteic acid was performed according to Moore ~6. Tryptophan was analyzed by the N-bromosuccinimide method ~7. Certain fragments were analyzed and compared by the technique of peptide mapping. Isolated polypeptides were treated with performic acid TM prior to tryptic digestion. Enzymatic cleavage was carried out in o.2 M NH4HCO a, p H 8, for 3 h at 37 °C at an enzyme :substrate ratio of 1:5 o. Trypsin, obtained from the Worthing-
HUMAN
IMMUNOGLOBULIN
M
449
ton Biochemical Corp., was reacted with L-(I-tosylamido-2-phenyl)ethyl chloromethyl ketone to inactivate chymotrypsin activity19. Digestion was stopped by adding an equivalent amount of soybean trypsin inhibitor (Sigma Chemical Co.). Chromatography was performed on Whatman No. 3 paper, using the solvent system : n-butanol-pyridine-acetic acid-water (15 :IO :3:12, by vol.) (see ref. 20). This was followed by high voltage electrophoresis in pyridine acetate buffer, pH 3.6 (see ref. 12) or in acetic acid-formic acid, pH 1. 9 (see ref. 21). For the identification of cysteine peptides, samples were completely reduced and alkylated using radioactive iodoacetamide at a specific activity of 8. 3/~Ci per mg of the sample 7. The peptides were detected autoradiographically. Qualitative N-terminal analysis was done by the dansyl method of Gray 2~ except that the dansyl amino acids were separated on a polyamide thin layer23. Hydrazinolysis was employed for C-terminal analysis according to Press et al. 24. Amino acid sequence of the C-terminal peptide of the # chain was determined by the dansyl-Edman technique as described by Gray 25. The sequence of the Nterminal portion of the K chain followed from analysis of the whole chain in the automatic sequenator 2~. RESULTS
CNBr fragments of partially S-sulfonated # chain CNBr split # chain was separated on Sephadex G-2oo in 5 M guanidine (Fig. I). Small peptides were more completely resolved using I M acetic acid with o.I M formic acid on Sephadex G-25. The latter also avoided difficulties with desalting. Unresolved large peptides were concentrated by pressure dialysis and recycled on Sephadex G-2oo in 5 M guanidine. Seven fractions were obtained and designated HA, HB, HC, HD, HE, HF, HG. Fraction HA corresponded to aggregates as determined by the elution volume and by peptide maps. The yield of Fraction HD was only 6% of the theoretical yield, as calculated from its estimated molecular weight. Under the conditions of low temperature cleavage this fraction was not detected, and therefore, was considered the product of a side reaction. Polyacrylamide disc electrophoresis in sodium dodecyl sulfate of Fractions HB and HC resulted in single bands with small quantities of aggregates. The HB and HC bands correspond to molecular weights of 35 ooo and 30 600, respectively. Weight E O
=, 0.4
I~
Ii Ii
Fig. I. S e p a r a t i o n of C N B r split/~ c h a i n (45 ° mg) on S e p h a d e x G-2oo c o l u m n (98 c m × 7.7 cm) in 5 M g u a n i d i n e . HCI. I n t h e t e x t t h e letter H precedes t h e d e s i g n a t i o n of fractions.
450
j . ZIKAN, J. c. BENNETT
C E
H
HclWr
Fig. 2. P o l y a c r y l a m i d e disc clectrophoresis of ¢* c h a i n (H) a n d C N B r s p l i t / , c h a i n (H c x Br) in o. ] M s o d i u m p h o s p h a t e (pH 7.2) with o. t % s o d i u m dodecyl sulfate.
average of molecular weights of H B and HC peptide determined by high speed short column sedimentation equilibrium were 46 IOO and 27 5oo, respectively. Fraction H E formed a single band corresponding to a molecular weight of 580o as determined by gel filtration (Fig. 2). However, in acid urea H E became a minimum two bands (Fig. 3a). Fractions H F and HG were not detected on the discs. After total oxidative sulfitolysis the molecular weights of Fractions H B and HC were lowered while the molecular weight of H E was unchanged. S-sulfonated H B fraction yielded three bands on sodium dodecyl sulfate gel electrophoresis corre-
a
b
HB HE
lib HB$ ~
HBB
Fig. 3- Polyacrylamide disc electrophoresis : (a) of C N B r split# (H) chain, H B and H E peptide in acid urea. (b) of Fraction H B , S-sulfonated H B , H B A and H B B (SE-Sephadex G-25 chromatography) in alkaline urea.
HUMAN
IMMUNOGLOBULIN
M
451
s p o n d i n g t o m o l e c u l a r w e i g h t s o f 29 ooo, 18 ooo a n d 14 5oo (Figs 3a a n d 3b). Ss u l f o n a t e d H C a p p e a r e d as a single b a n d w i t h a m o l e c u l a r w e i g h t o f 17 ooo. S - s u l f o n a t e d H B f r a c t i o n was d i v i d e d on an S E - S e p h a d e x c o l u m n (1.2 c m × 21 cm) in 8 M u r e a w i t h 5 m M NaC1 a d j u s t e d b y f o r m i c a c i d to p H 3.5- A f t e r e l u t i o n of t h e H B A f r a c t i o n a l i n e a r g r a d i e n t (250 m l o r i g i n a l buffer a n d 250 ml buffer w i t h 0.5 M NaC1) was b e g u n . O n gel e l e c t r o p h o r e s i s t h e H B B f r a c t i o n s h o w e d a single b a n d (mol. w t 14 500) in s o d i u m d o d e c y l s u l f a t e ; b u t in a l k a l i n e u r e a prod u c e d t w o b a n d s (Fig. 3b). H F a n d H G f r a c t i o n s w e r e e x a m i n e d a n d purified b y h i g h v o l t a g e p a p e r electrophoresis. H F f r a c t i o n g a v e t w o spots. H o w e v e r , t h e e l u t e d m a t e r i a l s g a v e i d e n t i c a l a m i n o a c i d c o m p o s i t i o n s , a n d t h e s e p a r a t i o n s w e r e c o n s i d e r e d as r e f l e c t i n g a m i d e differences. F r a c t i o n s H F a n d H G w e r e P a u l y r e a g e n t p o s i t i v e ; b u t o n l y F r a c t i o n H G was n i t r o p r u s s i d e p o s i t i v e . R a d i o a c t i v e p e p t i d e m a p s of t o t a l l y r e d u c e d a n d a l k a l a t e d (iodo[14C]acet amide) H B a n d H C p e p t i d e s s h o w e d s e v e n a n d six significant single h a l f c y s t e i n e peptides, respectively. N - t e r m i n a l a n a l y s i s of t h e f r a g m e n t s g a v e p h e n y l a l a n i n e a n d g l u t a m i c acid for H B , proline a n d v a l i n e for HC, g l u t a m i c acid for H E , g l u t a m i c a c i d for H F , a n d serine for H G . A n i n h y d r i n n e g a t i v e p e p t i d e in H B p e p t i d e m a p s s u g g e s t e d a b l o c k e d N - t e r m i n a l g r o u p . T y r o s i n e was f o u n d at t h e C - t e r m i n u s of t h e H G p e p t i d e . TABLE I A M I N O ACID C O M P O S I T I O N OF
Asp Thrt Ser~ Glu Homoserine Pro Gly Ala Val Met Ile Leu Tyr Phe Lys His Arg Trpt~ Cys§
CNBr
PIECES OF ~ CHAIN
B*
C*
E*
F*
G**
Total residues
i~ ***
18. 4 19.7 34.2 13.o 3.o 13.o 2o.o 15.8 21.6 o.o 5 .6 17. 5 5.1 l°.3 13.o 2.8 lO.4 3 .2 5.2
18.2 31.o 23.6 7.o 2.o 14.4 lO. 3 19.o 19.o o.o 7.9 16. 7 4.6 7.7 6.2 3.0 7.7 3.7 6.0
5.3 3.9 5.2 1.9 i.o 0.9 4.2 1. 7 4.0 o.o 2. 4 3.1 1. 7 0.9 2.9 o.8 59 i.i o.o
0.3 I.I 2.0 1. 7 i.o 3.1 1. 3 I.I 1.2 o.o o.I I.I I.O o.o I.O 0.9 0.9 o.o o.o
I.O 1. 5 I.O o.I o. i o.o I.O I.O o.o o.o o.i o.i I.O o.o o.o o.o o.i o.o 0.9
43.2 57.2 66.0 23-7tt 7-I 31.4 36.8 38.6 45.8 o.o 16.1 38.5 13. 4 18. 9 23.1 7.5 25.0 8.0 12.1
44.1 52.7 62. 3 52.2 o.o 34.7 41.4 33.0 45.6 7. I 15.8 39.2 13. 3 19.8 24. 5 8.7 26.2 9.2 12.1
* Related to proposed number of homoserine residues. ~* Related to one aspartic acid. *** Related to molecular weight of 65 ooo (Schrohenloher, R. E., unpublished) corrected for suggar content 2. t Corrected for losses during acid hydrolysis. t t The reaction of the ;u chain with CI~Br decreased the content of glutamic acid to 23.5 residues per molecule. The mechanism of this modification of glutamic acid will be further studied. t t t Analyzed by the N-bromosuccinimide method 1~. § Calculated as cysteic acid after performic acid oxidation TM.
452
J. ZIKAN, J. C. BENNETT
The amino acid compositions of CNBr fragments of the # chain are summarized in Table I and compared with the composition of the whole # chain. Compositions of Peptides H E and H F were related to a single homoserine. The composition of the multichain Peptide HB was related to three homoserines and of the Peptide HC to two homoserines. The validity of these relationships will be discussed. Peptide HG contained no homoserine and was sequenced by the dansylEdman method. Its structure was found to be: Met/Ser, Asp, Thr, Ala, Gly, Thr, Cys, Tvr.
CNBr fragments of partially S-sulfonated ~c chain CNBr split K chain was separated on Sephadex G-ioo in i M acetic acid and o.I M formic acid (Fig. 4a). Fractions were designated LA, LB, and LC. The Fraction LA appeared to be composed of aggregates by virtue of the value of the elution volume and by the peptide map which was similar to that of Fraction LB. The Fraction LB formed a band corresponding to a molecular weight of 21 ooo in sodium dodecyl sulfate gel electrophoresis. In alkaline urea gel electrophoresis, LB was found
g
I00
500
300
ml
IO0
300
~00
ml
rt
O00
300
-
5CO
rnl
I00
~
500
ml
Fig. 4. Chromatography of CNBr spTit proteins: (a) L(K) chain (28o mg), (b) F(c)5/~ (2o7 mg), Co) Fab l~ (27o mg), and (d) TgM (240 mg) on a Sephadex G - l o o column (86 cm × 2.5 cm) in I M acetic acid with o.i M formic acid. I n the t e x t the letter designating the whole molecule precedes the designation of fractions.
HUMAN IMMUNOGLOBULIN ~V[
453
to contain a fast moving component (J-chain) as does also the starting preparation of whole K chain. Peptide LC was not detected on disc electrophoresis, but was examined by paper high voltage electrophoresis. The amino acid compositions of Peptides LB and LC were compared with the whole K chain (Table II). The Peptide LB did not contain any homoserine and, therefore, its composition was related to the number of aspartic acid residues in the whole K chain, since the remainder of the K chain (LC) contained no aspartic acid residues. The composition of the LC peptide was related to one homoserine. End group analysis indicated that the N-terminal residues were glutamic acid in the LC peptide and threonine in the LB peptide. Sequencing of whole K chain with the automatic sequenator confirmed the primary structure of the LC peptide: Glu, Val, Ile, Met.
CNBr pieces of F(c)5 # fragment CNBr split F(c)5 # fragment was separated on Sephadex G-Ioo in I M acetic acid with o.i M formic acid (Fig. 4b). Fractions were designated FcA, FcB, FcC, and FcD. The yield of FcB peptide was low for its apparent molecular weight, but contained J-chain determinants2L FcA fraction resembled by molecular weight the whole F(c)5 # fragment. The Peptides FcC and FcD were undetectable on disc electrophoresis, but from paper electrophoresis, corresponded in mobility and composition to Peptides H F and HG, respectively. The tryptic peptide map and amino acid composition of the FcA peptide were similar to the Peptide HC (Fig. 5 and Table III). TABLE
[[
AMINO ACID COMPOSITION OF C N B r PIECES OF L CHAIN
Asp Thrt Sert Glu Homoserine Pro Gly Ala Val Met Ile Leu Tyr Phe Lys His
Arg Trpt t Cysttt * ** *** t ~t ttt
B*
C**
B+C
L***
17.o 18. 9 25.5 23. 5 o.o 12.1 14.1 17. 3 13. 9 o.o 6. 4 16.6 7.9 8.2 IO.I 2. 4 9.2
o.o o.o o.i 0. 4 I.O o.o o.1 o.o o. 9 o.o 0.8 o.o o.o o.o o.o o.o o.o
17.o 18.9 25.6 23. 9 i .o 12.1 14.2 17. 3 14.8 o.o 7.2 16.6 7.9 8.2 io.i 2.4 9.2
17.o 18.8 25.7 24. 9 o.o 11.9 13.9 16.2 14. 7 i.i 7.0 16. 7 7.8 8.0 9.7 2.3 8. 7 2. I 4.9
R e l a t e d t o 17 a s p a r f i c a c i d . Related to 1 homoserine. R e l a t e d t o m o l e c u l a r w e i g h t o f 22 5 o 0 (ref. 37). Corrected for losses during acid hydrolysis. Analyzed by the N-bromosuccinimide methodIL C a l c u l a t e d a s c y s t e i c a c i d a f t e r p e r f o r m i c a c i d o x i d a t i o n 1~.
Fig. 5- Peptide m a p s o f l , chain Peptides HB, HC and H E are c o m p a r e d with analogous peptides f r o m hot t r y p t i c f r a g m e n t s : FabB, F a b l ) and FcA. In the peptide m a p s of F a b B and H B, c o m m o n spots are circled; spots corresponding to I.B peptide are designated x. Letters v and g refer to yellow and grey colored spots, respectively.
HUMAN IMMUNOGLOBULIN M
TABLE
455
III
AMINO ACID COMPOSITION OF THE PEPTIDES F c A AND H C
FcA
Asp Thr*"
Ser *~ Glu Homoser Pro Gly Ala Val Ile Leu Tyr Phe Lys His Arg Trp~ Cystt
19 28.8 22.8 9.2 2.o 16. 4 lO. 4 19.1 20. 4 8.6 17. 7 5.6 8.0 5.8 4.o 7.7 3.6 5.9
*
HC*
18.2 31.o 23.6 7.o 2.o 14. 4 lO. 3 19.o 19.o 7.9 16. 7 4.6 7.7 6.2 3.o 7.7 3.7 6.0
* Related to two homoserines. ** Corrected for losses during acid hydrolysis. A n a l y z e d b y the N - b r o m o s u c c i n i m i d e methodlL t t Calculated as cysteic acid after performic acid oxidation le.
After oxidative sulfitolysis FcA peptide gave on polyacrylamide disc electrophoresis in sodium dodecyl sulfate three bands. Following separation on Sephadex G-2oo in 5 M guanidine, peptide maps showed that the third fraction (smallest peptide) was included in each of the first two fractions. It was considered to be derived from a spurious tryptic cleavage site.
CNBr pieces of Fab # fragment CNBr split Fab # fragment was separated on Sephadex G-Ioo in I M acetic acid and o.I M formic acid (Fig. 4c). Fractions were designated FabA, FabB, FabC, FabD, and FabE. According to molecular weight and peptide maps Fraction FabA was composed of aggregates, and the yield of Fraction FabC was insignificant. Gel electrophoretic behavior after oxidative sulfitolysis, peptide maps and amino acid composition indicated a similarity between the FabB fraction and the HB plus LB fractions and between the MB fraction isolated from whole IgM split with CNBr. However, the FabB has lower molecular weight (48 ooo) than the MB (54 900) or the HB plus LB (56 ooo) as determined by polyacrylamide disc electrophoresis. Besides, in peptide map of FabB it was possible to identify main spots corresponding to the LB peptide, however, we failed to find all spots corresponding to the HB (Fig. 5). Amino acid composition of the FabB was somewhat deficient as compared with compositions of the HB plus LB (Table IV). Totally S-sulfonated FabB fraction was separated on Sephadex G-Ioo in 5 M guanidine.HC1. In this manner aggregates were separated from a peptide which was similar to the Fraction HBB by gel electrophoretic behavior and amino acid composition.
456
j. ZIKAN,
TABLE
J. c. BENNETT
IV
AMINO ACID COMPOSITION OF T H E P E P T I D E F a b B PEPTIDE
Asp Thr*** Ser***
Glu Homoser
Pro Gly Ala
Val Met Ile Leu Tyr Phe Lys His Arg Cys~
FabB*
L B + HB*"
33.4 27.6 58.5 t9.6 2.o 21.3 33.o 33. ,t 34 .o -lO.2 31.0 lO.2 14.1 17.7 5-9 18. 3 lO.O
35.4 38.0 59.7 36.5 3.o 25.1 34.1 33. I 35.5 -12.o 34.2 i3.o 18. 5 23.2 5.2 19.6 IO.I
AND THE SUM OF COMPOSITIONS OF E l { AND H g
* Related to t w o h o m o s e r i n e residues. ** R e l a t e d to three h o m o s e r i n e residues. "** Corrected for losses during acid analysis. t Calculated as cysteic acid after performic acid o x i d a t i o n 16.
An Fd-like peptide was produced from Fab by partial oxidative sulfitolysis and separation from L chain on Sephadex G-Ioo in 0.2 M acetic acid-o.oI5 M NaC1. Reaction o f this Fd component with CNBr released a fraction identical to FabB, but without LB peptides. The Fraction FabD was similar to the Fraction HE by gel electrophoretic behavior, peptide map and amino acid composition (Fig. 5 and Table V). The Fraction FabE, which was not detectable on disc electrophoresis was identified by paper electrophoresis as a mixture of small peptides, including the LC peptide. CNBr
pieces of whole IgM
Small CNBr pieces of IgM were separated on Sephadex G-Ioo ill I M acetic acid with o.I M formic acid (Fig. 4d). Larger CNBr pieces in the first fraction were concentrated by pressure dialysis and separated further on Sephadex G-2oo in the same solvent or in 5 M guadinine. Fractions were designated MA, MB, MC and MD. Polyacrylamide disc electrophoresis and peptide maps showed that the Fraction MB was related to the Peptide FabB and that the Fraction MA was related to the FcA peptide and also aggregated Fraction MB (Fig. 6). The Fraction MC by peptide maps and gel electrophoresis was identified as the HE peptide. The Fraction MD contained small peptides corresponding to LC, HF and HG. DISCUSSION
This paper presents a comparison of the CNBr pieces produced from IgM (Dau),
HUMAN IMMUNOGLOBULIN M
457
TABLE V A M I N O A C I D C O M P O S I T I O N OF T H E P E P T I D E H E
AND THE PEPTIDE
FabD
C o m p a r i s o n w i t h t h e p e p t i d e F2 r e p o r t e d b y W i k l e r et al. 85.
HE* Asp Thr*** Ser*** Glu Homoser Pro Gly Ala Val Ile Leu Tyr Phe Lys His Arg Trp f Total
5-3 3.9 5 .2 1.9 i.o 0. 9 4 .2 1.7 4 .0 2. 4 3.1 1.7 0.9 2.9 0.8 5.9 i. i 46.9
FabD* 5-3 4 .0 5 .2 1.8 i.o 1.2 3.7 2. I 3 .6 2.0 3.1 1.7 1-3 2.6 0.8 4.6 i.o 44.9
F2** 7 2 4 2 i 2 i 2 3 I 4 5 I I 4 5 3 47
* R e l a t e d to i homoserine. ** C a l c u l a t e d f r o m s e q u e n c e of p e p t i d e F2. *** C o r r e c t e d for losses d u r i n g acid h y d r o l y s i s . t A n a l y z e d b y t h e N - b r o m o s u c c i n i m i d e methodZL
Fig. 6. P o l y a c r y l a m i d e disc e l e c t r o p h o r e s i s of S - s u l f o n a t e d F r a c t i o n s MA, MB, F c A a n d F a b B in a l k a l i n e urea.
458
J. ZIK/N, J. C. BENNETT
i t s / , a n d K chain, a n d its F(c)a u a n d F a b # f r a g m e n t s . P r e l i m i n a r y o r d e r i n g of t h e C N B r pieces w i t h i n this I g M m o l e c u l e is a f f o r d e d b y t h e s e d a t a (refer to F i g s 7 a n d 8). R e s u l t s f r o m t h e K c h a i n are c o n s i s t e n t w i t h t h e p r e s e n c e of one m e t h i o n i n e residue. T w o C N B r pieces were i s o l a t e d , L B a n d L C T h e L B p e p t i d e did not c o n t a i n h o m o s e r i n e a n d is, t h e r e f o r e , C - t e r m i n a l w h i l e t h e LC p e p t i d e is N - t e r n f i n a l . T h e s e d a t a are c o m p a t i b l e w i t h p r e s e n t k n o w l e d g e of K-chain s e q u e n c e 2~.
ISOLATION
IgM
OF IgM (Dau) CNBr FRAGMENTS
H-Choin(So 2-)
IqM
a g MA
MB
HA,HB,HC + peptides
MC
1,3,4
5,7
1,3,4
o~
2
5,7
HE,HF,HG
LB, J
F(c)sp
Fob
2
FcA, FcB,FcC, FcD
FobB, FobO, FobE
6 8 1,3,4,kO 2
peptides
5,7
d
6
8
Fig. 7. Isolation of IgM(Dau) CNBr fragments. Flow sheet to summarize the various procedures to isolate and derive the relative order of the CNBr fragments of IgM (Dau). The numbers refer to the placement order from the N-terminus of the/~ chain. S # S refers to cleavage of disulfide bonds.
HL
I
I -1
HI HI
1 • I..l
II
__
i •
Ht I
2
3
5
4
6
7
8
HI H2 0--- CHO
Fig. 8. Order of IgM H chain CNBr fragments. Scheme of the arrangement of CNBr pieces and disulfide bonds of IgM (Dau). The half eysteine residue involved in the interehain bridge between different 7-S subunits is designated H1H=, and those between # chains inside one 7-S subunit, H1H 1. The arrow designates the site of trypsin attack. The closed circles represent the location of carbohydrate peptides.
HUMAN IMMUNOGLOBULINM
459
A more complicated situation exists with the CNBr pieces of the/~ chain. These difficulties arise from the following ambiguities: i. Some single CNBr pieces were not isolated due to difficulties with their separation. 2. As a result of the uncertainty in determination of molecular weights especiaUy of # chain and of the Peptide HB, the data are not conclusive as to whether the latter fragment contains three or four methionine residues. The amino acid data were calculated using the molecular weight of # chain (Dau) as 65 ooo as was determined by sedimentation equilibrium in guanidine (Schrohenloher, R. E., unpublished). Previous determinations on this molecule have resulted in /,-chain weights of 80 ooo from sedimentation data 29 and 75 ooo from gel filtration s°. Best agreement with # chain composition was found when the composition of HB peptide was related to three homoserine residues. In the HB peptide two N-terminal groups were found. A third blocked Nterminal group was also present 4. However, the molecular weight of the HB peptide from sedimentation equilibrium data (46 IOO) and the number of half cysteine peptides (seven) by autoradiography were consistent with up to four CNBr pieces. This high average molecular weight could be explained by the presence of aggregates in the HB peptide fraction. This high number of half cysteine peptides could correspond to heterogeneity in a glycopeptide and give rise to an erroneous result, i.e. two spots in the peptide map could correspond to the same part of the peptide chain. Contrarywise, one disulfide bridge could be less accessible for modification and the number of analyzed cysteic acid residues might be too low. This possibility deserves more consideration since five intrachain disulfide bridges have been found in another myeloma IgM (ref. 31). The assumption, however, has been that the extra S-S loop is in the Fc portion of the molecule3~. Further experimentation, which is now underway, is needed to clarify this point. After oxidative sulfitolysis two fragments from the HB peptide (mol. wts 14 500 and 18 ooo, HBB and HBA, respectively) were derived and were identified by gel electrophoresis in sodium dodecyl sulfate. The third fragment composing the HB peptide is smaller and has the blocked N-terminus. By means of chromatography on SE-Sephadex G-25 two fractions (HBA, HBB) were separated which differed in electrophoretic mobility in alkaline urea, in peptide maps and amino acid composition. From this it was concluded that single CNBr pieces (designated I, 3 and 4 in Fig. 8) were connected in HB peptide by disulfide bonds. In the peptide map of FabB peptide it was possible to identify the major LB peptides, however, all spots corresponding to the HB peptide were not found (Fig. 6 and Table IV). Similarity of FabB and HB peptides is evident also from data concerning the carbohydrate content2m and from gel electrophoresis. Therefore, the HB peptide must belong to the Fd part of the # chain and the trypsin susceptible peptide bond is located toward the C-terminal part of this peptide. The presence of a peptide with a blocked N-terminal group is an additional reason for placing HB in this part of the chain (Fig. 8). The Peptide HBB (mol. wt 14 500) was released also from S-sulfonated FabB peptide. Therefore, the HBB peptide was not located in that part of the peptide HB which is susceptible to attack by trypsin, and must be placed in the middle of the HB peptide (designated 3 in Fig. 8). HC peptide (designated 5 and 7 in Fig. 8) contained two N-terminal amino
460
J. ZIKJ~N, J. c. BENNETT
acids and after oxidative sulfitolysis it could be resolved into two pieces on Sephadex G-Ioo in 5 M guanidine (Bertolini, M., Hurst, M. and Bennett, J. C., unpublished). The smaller piece is similar to the region located by Putnam el al. 34 toward the Cterminus of the chain. HC peptide was similar to the l;cA peptide (1;ig. 5 and Table III). According to peptide maps, amino acid compositions and electrophoretic behavior H E peptide (designated 2 in Fig. 8) was similar to the FabI) peptide and resembled in size and amino acid composition the 1;2 peptide reported by Wikler at al. a5 (Table V). By analogy a5 it probably occupies a second position in the order of CNBr pieces of the # chain. H F peptide was found also in CNBr pieces of the F(c)5 # fragment. Amino acid sequence of H F corresponded to the FY peptide reported by Wikler ,;t al. 35 and is homologous with part of rabbit Fc fragment in Position lO8-122 as reported by Hill et al. ~6. Accordingly H F is probably located between the two pieces of the H(" peptide (designated 6 in Fig. 8). HG peptide, released from/z chain and the F(c)a# fragment, did not contain homoserine, but had C-terminal tyrosine, as was also found for the intact # chain. Amino acid sequence of HG is identical to the FZ peptide of Wikler el a/. '~r', and is considered to be C-terminal (designated 8 in Fig. 8). Following oxidative sulfitolysis of the FcA peptide its approximate molecular weight was reduced to nearly o.o 5 of the original weight. FcA, therefore, probably contains interchain disulfide bonds responsible for keeping the subunits together. On the basis of current models for IgM it is likely that at least one of these interchain disulfide bonds connects individual 7-S subunits, and a second one connects # chains within one subunit. The C-terminal peptide HG must connect # chains only within one subunit because it was released from CNBr split F(c)5 # fragment and IgM as a low molecular component without disruption of disulfide bonds. The probable arrangement of disulfide bonds derived from the above data is included in Fig. 8. When comparing the partial structure of IgM (Ou) a~ with our order of CNBr pieces of IgM (Dau), several additional relationslfips are of note. The H E peptide (designated 2 in Fig. 8) and analogous peptide of IgM (Ou) including the subgroupspecific region have different compositions. Since a part of the Peptide HB (3,4) includes the hypervariable region, differences including the number of methionine residues are not unexpected. The identity of the HI" (6) and the HG (8) peptides with the analogous peptides of IgM (Ou) suggests similarity of the C-terminal domains of both ~, chains. ACKNOWLEDGMENTS
We thank Mr Tom Stanton and Mr Danny Lynn for analysis of primary structure, Miss Freda T. Moore for help with amino acid analysis and Dr Eduard J. Moticka for helpful discussion. This work was supported by the John A. Hartford l;oundation, Inc.
HUMAN
IMMUNOGLOBULIN
M
461
REFERENCES I 2 3 4 5 6 7 8 9 io ii 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
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