Identification of a Third Type of λLight Chain in Mouse ImmunoglobulinsAuthor(s): Takachika Azuma, Lisa A. Steiner and Herman N. EisenSource: Proceedings of the National Academy of Sciences of the United States of America,Vol. 78, No. 1, [Part 2: Biological Sciences] (Jan., 1981), pp. 569-573Published by: National Academy of SciencesStable URL: http://www.jstor.org/stable/10087 .

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Proc. Natl. Acad. Sci. USA Vol. 78, No. 1, pp. 569-573, January 1981 Immunology

Identilication of a third type of A light chain in mouse immunoglobulins

(myeloma proteins/monoclonal antibodies/immunoglobulin genes)

TAKACHIKA AZUMA*, LISA A. STEINER, AND HERMAN N. EISENt

Department of Biology and Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

Contributed by, H. N. Eisen, October 16, 1980

ABSTRACT A third type of mouse A chain was identified in the course of examining light chains from four myeloma proteins and two monoclonal antibodies. On the basis of their antigenic prop- erties and, a characteristic COOH-terminal tryptic peptide, the light chains from these immunoglobulins had previously been pro- visionally identified as A2 chains. However, four of these chains, designated now as A3 (or Al,,), were found to share constant region features that clearly distinguish them from the other types of mouse A chains (Al and A2). The amino -acid sequence of more than 90% of the A3 constant region (from.position 120 to the COOH-terminus) was: determined. In this region the A3 sequence differs from the A2 constant region at 5 positions and from, the Al constant region at about 30 positions.

About 95% of immunoglobulins in the mouse have light chains of the K type (1, 2). The first indication that A light chains in the remaining 5% have diverse forms was provided by the amino acid sequence of the light chain (L315) of myeloma protein MOPC-315 (3, 4): .the. amino acid sequence of the constant re- gion of this.chain differed at about 30 of l10 positions from the constant region of other A chains, represented by L'04, the light chain of myeloma protein MOPC-104E (5). Chains with the con- stant region of L104 are called A1 (or A,); those with the constant region of L315 are called A2 (or All) (3). Analyses of antigenic properties and of the COOH-terminal peptides obtained by tryptic digestion of light chains showed that in most strains of mice about 1% 'of normal serum, immunoglobulins have A2 chains and about 4% have, Al chains (2, 6). We show here that some light chains previously considered to be A2 are another type of A chain, which we designate as, A3 (or AIII).

We first recognized A3 chains through a chance observation in the course of evaluating the. difference in amino acid se- quence between'L315 (the prototype A2.chain) and the sequence encoded by the germ-line gene for the A2 variable region (7). For this evaluation.we examined.six. chains that had previously been provisionally identified as A2 chains (unpublished data). When the fragments produced by cleaving the chains with CNBr were analyzed we found that four of them,. the A3.chains, differed from L3 5. Analysis of the sequences of'fragments com- prising 90% of the constant region demonstrated that A3 chains differ from A2 at 5 positions and from Al chains at about 30 po- sitions.

MATERIALS AND METHODS

Immunoglobulins. Myeloma tumors were obtained from Mi- chael Potter (National Institutes of'Health) or from Litton Bio- netics (Baltimore, MD) and were maintained'by serial passage in. B:ALB/c AnN. mice. Myeloma proteins CBPC-49, ABPC-72, and TEPC-952 were isolated by chromatography on QAE- Sephadex A-5 and'by gel'filtration.on Sephadex G-200 from. as-

The publication costs of this article were de-frayed in part by page charge payment. This article must therefore be hereby* marked "advertise- ment" in accordance with 18 U. S. C., ?1734 solely to indicate this fact.

cites fluid or serum of tumor-bearing mice (8).. Protein SAPC-15 was isolated in the same way from ascites fluid kindly supplied by M. Potter. Hybridomas 8-13. and 8-47, produced .by fusing spleen cells from BALB/c mice (immunized with 2,4,6-trinitro- phenylhemocyanin) with NS-1 myeloma cells, were generously .provided by Ann Marshak-Rothstein and Malcolm Gefter. The monoclonal antibodies secreted by these. hybridomas (and also by myeloma MOPC-315) were isolated from ascites fluid by ad- sorption to dinitrophenyllysyl-Sepharose and elution with dini- trophenylglycine (9).

Light Chains. The purified immunoglobulins were partially reduced in 0..01 M dithiothreitol/0.2 M Tris HCl, pH 8.2; they were then alkylated with iodoacetamide and subjected to gel filtration on Sephadex G-100 in 6 M urea/I M acetic acid to separate heavy and light chains (10). To separate the A chains from contaminating K chains, the pooled light chains were sub- jected to gel'filtration on Sephadex G-75 in 0.,15 M NaCl/K 'phosphate, pH 7.;2 (PJ/NaCl). -Under these conditions, A chains form noncovalently associated dimers whereas most K chains .do-not (11, 12). The.removal of'K contaminants was especially important with light chains from the monoclonal (hybridoma) antibodies because many of -these molecules had.both A and K

chains; the latter were derived from the'fused myeloma NS-1 cells. Extensive reduction of the purified A chains (to cleave intrachain disulfide.bridges) was usually carried out with 0.01 ,M dithiothreitol/6 M guanidinellCl/0.2 M Tris'HCl, pH 7.6, followed by alkylation with 4-vinylpyridine (13).

Chemical Modifications. Cleavage.of reduced or unreduced light chains at methionyl residues was carried out in'70% formic acid with a 150-fold molar excess of CNBr (with respect to me- thionine) at room temperature for 24 hr. Reaction with citra- conic anhydride was carried out as described (14) and the same ,method was used.for.succinylation. The procedure for treatment with hydroxylamine was as described (15). Ethyleneimine (ICN Pharmaceuticals, Plainview, NJ) was used as described (4) to alk- ylate citraconylated, reduced L49.

Automated Sequence Analysis. Edman degradation was per- formed in a Beckman 890 C sequencer with a 0.1 M Quadrol program .(16) and 6 mg of Polybrene. Phenylthiohydantoin (>PhNCS) amino acids were identified by gas/liquid chro- matography, high-performance liquid chromatography, thin- layer chromatography, and amino acid analysis after hydrolysis

Abbreviations: L315 L', L49 L72 and L'5 light chains of myeloma .proteins MOPC-315, MOPC-104E, CBPC49,.ABPC-72, and SAPC- 15, respectively; L813 and L8-47, A light chains of 8-13 and 8-47, two anti-Tnp monoclonal antibodies; P,/NaCl, phosphate-buffered saline (0.15 M NaCl/K phosphate, pH 7.2); >PhNCS, phenylthiohydantoin; VK, CK, VA, and CA, variable and constant regions of K; and A chains, respectively. * Present address: Faculty of Science, Department of Biology, Osaka ..University, Osaka, Japan. t To whom reprint requests should be addressed.

569

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570 Immunology: Azuma et al. Proc. Natl. Acad.. Sci. USA 78 (1981)

1.0

0.8 _ 8-13(A2) A

0.6

0.4-

0.2 Fr I

I Fr.I ~~~~~~F.II

B 0.8 - 8-47(A3)

0.6-

0.4-

Fr.l FrI 0.2 -

20 30 40 50 60 70 80

Tube

FIG. 1. Elution profile of CNBr fragments of a A2 (A) and a A3 (B) light chain (from hybridomas 8-13 and 8-47, respectively). Light chain (10 mg) was cleaved with CNBr and subjected to gel filtration on Seph- adex G-100 (1.6 x 90 cm) in 6 M urea/l M.acetic' acid at a flow rate of 10 ml/hr; 2 ml was collected per tube. Fr. I consisted of various partially cleaved chains.

with HI. (14). A standard for S-pyridylethylcysteine was pre- pared by treating S-/3-4-pyridylethyl-L-cysteine (Pierce) with a 10-fold molar excess of phenylisothiocyanate in 50% pyridine for 1 hr under N2 at 40?C. After thorough extraction with ben- zene, the aqueous phase.was dried and treated with 1 M HCI for 10 min at 80?C, followed by extraction with ethyl; acetate; hydrolysis of the aqueous. phase yielded alanine.

Amino Acid Analysis., Procedures for hydrolysis and for amino. acid analysis with.a Durrum D500 analyzer have been de- scribed (14). Under the conditions used, S-pyridylethylcysteine was eluted as a broad peak. after NH3 but we were unable to measure it, accurately.

Micro .Dansyl Procedure. This was based on- a modification (17) of the procedure described-by Hartley (18).

RESULTS

Cleavage of L315 at its two methionine residues yields three frag- ments. As shown (4), two of these are similar in size and, on gel filtration. with Sephadex. G-100, migrate. in the same fraction (Fig. 1A, Fr. IL); the third fragment is more retarded (Fig. IA; Fr. ILL). The other A chains examined in this study also contain two residues of methioninet (8) and would also be expected to. yield three fragments after cleavage. with CNBr. In the case of chains L.52 and L8'-3, the three fragments were distributed upon gel filtration exactly as were those from L315 (Fig. 1A). How- ever, the fragments. obtained from four.other chains (L49, L72, L847, and L'5) were distributed differently,.as shown by the representative pattern in Fig. 1B; the properties of the CNBr cleavage -products from these four- chains are described below.

t L15 actually contained 2.5 methionines per 214 residues, but the ad- ditional methionine could have been due to the unusually high K chain contamination of.the L15 preparation analyzed.

0.4

0.3 Fr. III

0.2

0.1

0.3

CB-IIIA

0.2

_0.1

/0 .. 1

0.3

CB-IIIB'

0.2-

0.1

60 70 80 90 100 110

Tube

FIG. 2. Resolution of Fr. III. of L8-47 (see Fig. iB) into fragments CB-IIIA and.CB-IIIB. Fr. III was -dialyzed against 1 M acetic acid, ly- ophilized, redissolved.in 1.5 ml of 1 M acetic acid, and.subjected to gel filtration on Sephadex G-50 (1.2 x 110 cm) in 1 M acetic acid (flow rate, 3 ml/hr; volume per tube, 1.2 ml). The incompletely separated frag- ments, shown in the top panel were rechromatographed. on the same column. The hatched areas indicate the tubes that.were pooled for fur- ther characterization of the fragments.-

No a-NH2 group could, be demonstrated in the material in Fr. IL (Fig. 1B). Because all mouse A chains examined so far have a blocked NH2 terminus (19), it was presumed that Fr. II contains the NH2-terminal CNBr fragment, designated CB-LI. In contrast, two NH2-terminal residues, phenylalanyl and alanyl, were found in Fr. III (Fig. IB); the components in this pool were resolved by gel filtration with Sephadex G-50 into two fragments, CB-IIIA and CB-IIIB (Fig. 2). Homoserine was present in CB-I and CB-IIIA but was absent from CB-ILLB; therefore, CB-IIIB must be the COOH-terminal fragment. The NH2-terminal sequence of fragment CB-IIIA (of L49) was Phe- Pro-Pro-Ser, a highly conserved sequence in all light chains, be- ginning at position 121? (19), suggesting that, in.this light chain, methionine replaces the valine at position 120 in L3 5. Fur- thermore, the methionine at position 87 in L315 appears to be absent from L49, but the methionine at 175 is conserved. Ev- idently, L49 and the other light chains in this set (L8-47, L72, L'5) belong to a new class of A chains (A3).

The location of methionine residues differentiates A3 from Al and A2 chains (Fig.3). These chains can easily be distinguished by subjecting their CNBr digests to electrophoresis in poly- acrylamide gels in the presence of sodium dodecyl sulfate. The differences are illustrated with A2 and A3 chains in Fig. 4.

? The numbering is that of L315 (4).

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Immonology: Azuma et al. Proc. Natl. Acad. Sci. USA 78 (1981) 571

vx ~~~cx

R I E 23 SS--~ 4 -i M-Mi* '-- Ser- COOH X i L 23 90 ~~~~137 162 175 195

)\2 [ '~~A Leu- COOH 87 I 175

X3 L VW ,

Leu--COOH 1 120 175

FIG. 3. Diagram of methionine residues in Al, A2, and A3 chains. The positions of these residues in Al and A2 chains are based on refs. 4 and 5; the positions of half-cystines in the variable regions of A3 are assumed to be the same as in the variable regions of Al and A2 (4,5,20).

The amino acid compositions of fragments CB-IIIA and CB- IIIB from two A3 chains (L49 and L847) and the corresponding regions or fragments of A2 chains are compared in Tables 1 and 2, and the sequences of A2 and A3 chains are compared in Fig. 5. Details on which these sequences are based are given in the Appendix.

The COOH-terminal fragments (176-214) of A3 and A2 chains were indistinguishable both in amino acid composition and in sequence. In contrast, the composition of CB-IIIA from L49 and L8-47 (Table 1) suggests that there are four amino acid substitutions between A2 and A3 chains from residues 120 to 175. These substitutions were explicitly established by analyz- ing the sequence of CB-IIIA and of three peptides derived from it: Ti and T2 obtained by tryptic cleavage of S-aminoethylated and citraconylated CB-IIIA from L49 and a peptide obtained by NH2OH cleavage of succinylated CB-IIIA from L847.

As anticipated, the sequence of peptide Ti corresponded to the NH2-terminal sequence of fragment CB-IIIA and the se- quence of the first 18 residues of T2 corresponded to positions 138-155 (Fig. 5). However, in the analyses of both intact CB- IIIA and peptide T2, the yield of >PhNCS amino acids de- creased markedly after glycine-155, and the remainder of the

cq UIZ co e.-

u y-4 0

1-87 - 1-120

88175 121-175

176-214 -_ < +721 176-214 -~ ~ ~~~~7621

A2 A3

FIG. 4. Electrophoresis of CNBr digests of two A2 and two A3 chains in polyacrylamide gel. A slab gel modification of the urea/so- dium dodecylsulfate system of Swank and Munkres (21) was used. The separating gel contained 15% acrylamide. The prominent slowly mov- ing band in digests of A3 chains is fragment CB-II, indicated provision- ally as spanning positions 1-120; the faster-moving band contains fragments CB-HIA (positions 121-175) and CB-IIIB (positions 176-214). The same pattern was obtained with digests of L72 and L15. With the A2 digests, the slower-moving prominent band contains two fragments (1-87 and 88-175) and the faster band contains the COOH-terminal fragment (176-214). Digests of A1 (not shown) also yieldtwo prominent bands: the slow one (migrating more slowly than the prominent slow band of A3 chains) encompasses positions 1-162 and the fast one, the COOH-terminal fragment (176-214).

Table 1. Amino acid composition of A2 and A3 chains at positions 121-175

A3 Fragment CB-IIA

Amino from acid A2* L49 L8-47

Asp 6 7.0 6.9 Thr 6 6.2 6.2 Ser 6 5.3 5.4 Glu 5 5.9 5.9 Pro 5 5.8 5.7 Gly 4 3.1 3.1 Ala 3 3.0 3.1 Val 4 3.9 3.9 Mett 1 0.9 0.8 Ile 2 1.9 1.8 Leu 3 3.1 3.1 Tyr 0 1.0 0.9 Phe 3 2.0 2.0 His 0 0.2 0.2 Lys 5 3.9 3.9 Arg 0 0.2 0.1 Trp 1 ND ND Cyst 1 ND ND

Samples were hydrolyzed in constant boiling HCl for 24 and 72 hr. Val and Ie were based on 72-hr hydrolysis; Thr and Ser were deter- mined by logarithmic extrapolation of 24- and 72-hr values to zero time. Values in boldface differ significantly between L315 and the A3 chains. ND, not determined. * From the known sequence of L315 (4). t Methionine was determined as homoserine. t The cysteine residues in CB-IIA were substituted with 4-vinylpyr-

idine and were not determined by amino acid analysis.

sequence of CB-IIIA could not be determined. Presumably, this decrease was due to a rearrangement at the Asn-Gly se- quence at 154-155 (for discussion, see ref. 14). Accordingly, to cleave after the asparginyl residue, fragment CB-IIIA was treated with hydroxylamine after it had been treated with suc- cinic anhydride to block the a-NH2 terminal group. The treated fragment was applied to the sequenator and, as expected, a sin- gle sequence, beginning at Gly-155, was obtained (Fig. 5).

The A2 and A3 chains clearly differ at five positions between residues 120 and 175 (at 120, 125, 129, 171, and 174). These dif- ferences agree with the amino acid composition data shown in Tables 1 and 2. The A3 sequence at positions 145 and 146 is Ser- Gly instead of Gly-Ser reported at these positions in L3"5 (4). This could represent another pair of differences between A2 and A3 constant regions, or it could reflect an error in the reported sequence of L3 5.

DISCUSSION In this study we examined six light chains that, on the basis of antigenic properties and a characteristic COOH-terminal tryp- tic peptide, were previously considered to have the same con- stant region as L315 and were thus identified as A2 chains (2, 6, 8). Based on cleavages at methionine residues, four of these chains (L49 L8-47, L72, and L'5), designated as A3 chains, were found to share distinctive constant region features that differ- entiate them from A2 and Al, the other types of A chains. Amino acid sequences of fragments of L49 and L847 showed that, be- ginning at position 120, the constant region of A3 chains differs in many more positions from the constant region of Al chains than from the constant region of A2 chains (approximately 30 and 5 differences, respectively).

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572 Immunology: Azuma et al. Proc. Natl. Acad. Sci. USA 78 (1981)

Table 2. Amino acid composition of A2 and A3 chains at positions 176-214

A2 A3

Expected Found in CB-III Found in CB-III B Amino from fragment from fragment from

acid L315 (4) L315 L952 L49 L8-47

Asp 3 3.1 3.1 3.2 3.2 Thr 4 4.0 4.2 4.1 4.1 Ser 7 6.8 6.9 7.0 6.8 Glu 5 4.7 4.6 4.7 4.8 Pro 1 1.0 1.1 1.1 1.2 Gly 1 1.3 1.6 1.3 1.4 Ala 2 2.2 2.1 2.0 2.1 Val 2 2.2 2.1 2.1 2.1 Met 0 - - - -

Ile 0 0.1 0.1 - 0.1 Leu 4 3.9 3.8 3.8 3.7 Tyr 0 0.1 - - -

Phe 2 1.9 1.9 1.9 1.9 His 3 2.7 2.6 2.7 2.7 Lys 1 1.1 1.1 1.2 1.1 Arg 1 1.1 1.0 1.0 0.9 Trp 1 ND ND ND ND Cys

CM-Cys 1 ND 0.7 0.6 0.7 Pe-Cys 1 ND ND ND ND

See footnotes for Table 1. -, <0.1; ND, not determined. Except for L3"5, after partial reduction the chains were alkylated with iodoacet- amide and after complete reduction they were alkylated with 4-vinyl- pyridine. For L315, 4-vinylpyridine was used for both alkylation steps. CM-Cys, S-carboxymethylcysteine; Pe-Cys, S-pyridylethylcysteine.

Because A3 chains are made by cells from the same inbred mouse strain (BALB/c) that produces Al and A2 chains, the A3 constant region must be another isotype, not an allelic variant of Al or A2 constant regions. At the DNA level, every immuno- globulin isotype is specified by a distinct germ-line gene. Thus, there must be three genes for A constant regions in the mouse. Those for Al and A2 (CA1 and CA2) have been detected by nucleic acid hybridization (22, 23) and one (CA1) has been cloned and its sequence has been determined (24, ?). Are there more than three genes for mouse A constant regions? There might well be more than three in view of the many constant region var- iants of human A chains. In humans, three distinct A isotypes

have been recognized (25-28) and several additional A variants, differing from each other at one or two scattered positions (29-31), have also been described. Whether the latter repre- sent somatic cell variants, allelic variants, or additional isotypes is not yet clear.

The expression of A genes in the mouse differs considerably from that of K and heavy genes. In the K family there seem to be many hundreds of variable region genes and most (or all) of them can probably be expressed in association with the single CK gene. In the heavy chain family there are also many hundreds (if not thousands) of VH genes and it is probable that they can be expressed with any of the CH genes (e.g., C,., C., etc.). In the much smaller A family, there seem to be only two variable region genes, VA] and VA2 (23). The variable regions of about 20 Al chains appear to be derived from the VA1 gene (24, 32) and the variable regions of three A2 chains appear to be derived from the VA2 gene.ll Thus, the A chains examined so far form two distinct groups according to their amino acid sequences. It is especially notable that, in contrast to the K and heavy chain fam- ilies, each VA gene appears to be expressed only in association with a particular CA gene.

Recent studies of immunoglobulin gene rearrangements in myeloma cells suggest that a critical step in the commitment of a B lymphocyte to produce a particular immunoglobulin chain is the approximation of V and C encoding sequences that, in the uncommitted cell, are many kilobases apart on the same chromosome (33-35). Hence, the consistent coexpression of VA1 with CA1 and of VA2 with CA2 would be readily understood if the V and C genes for Al were on a different chromosome than those for A2. However, a recent study of DNA from ham- ster-mouse somatic cell hybrids by nucleic acid hybridization with Al and A2 probes has shown that all of these A encoding sequences are on the same chromosome, no. 16 (ref. 22).

Fragmentary evidence suggests that there are less-stringent but perhaps similar restrictions in the expression of A3 chains. Thus, the ligand-binding activities of heavy-light chain recom- binant molecules in which Al, A2, and A3 chains were combined with a particular heavy chain (unpublished data) are consistent with the view that A2 and A3 chains have very similar V regions. Moreover, VA1 and VA2 genes have been shown by DNA se- quence data to be similar (7, 24). Hence, if a VA3 gene exists it should have hybridized, as did the VA1 and VA2 genes, with nucleic acid probes containing VAI sequences (23). That a third gene was not detected suggests that there indeed may be only two such genes, VA, and VA2, and that one or the other or both

1 A. L. M. Bothwell, M. Paskind, R. C. Schwartz, G. E. Sonnenschein, M. L. Gefter, and D. Baltimore, personal communication.

11 Azuma, T., Steiner, L. A. & Eisen, H. N. (1980) Fourth International Congress of Immunology, Paris, 1.1.01 (abstr.).

1 1 1 1 1 1 2 3 4 5 6 7 0 0 0 0 0 0

53 M F P P S P E E L Q E N K A T L V C L I S N F S P S G V T V A W K A N G T P I T Q G V B T S B P T K E * B (B,K) Y M

52 V S K G S (-) ( ) G N * K F-

I1 L S T T T-T D-Y-GV D-VD D - V ME-TE-S-QS N N

1 1 2 2 8 9 0 1 0 0 0 0

53 A S S F L H L T S D O W R S H N S F T C Q V T H E G D T V E K S L S P A E C L

52

X1 Y-T - R A W E S Y SS S (-Z-H-)--Q R-D-S

FIG. 5. Amino acid sequence for 95 of 110 residues of the A3 constant region in comparison with sequences for corresponding regions of A2 and Al chains. - , Same residue as A3; *, gap (to align A3 and A2 with Al, which has an extra residue between positions 171 and 173). The one-letter code is used: A, Ala; B, Asx; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; Y, Tyr. Details on which the sequences are based are given in the Appendix. See also Note Added in Proof.

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Immunology: Azuma et al. Proc. Natl. Acad. Sci. USA 78 (1981) 573

can be expressed in chains with the constant region of A3. This possibility is in accord with preliminary evidence that the CA3 gene is also on chromosome 16 (22). It is possible that the basis for V-C restriction in A chain expression will be clarified when the gene order (e.g., Vi, V2, ..., Ci, C2, C3 or Vi, Ci, ..., V2, C2, ..., etc.) on chromosome 16 is established and when the precise mechanisms responsible for joining V- and C-encoding sequences are known. Information on the joining mechanisms may also help to explain the marked differences in frequency of expression of the various mouse A isotypes: e.g., Al chains are about 5 times more frequent in normal serum immuno- globulins and in immunoglobulins elicited by various antigenic and mitogenic stimuli than the sum of A2 and A3 chains (ref. 2; unpublished data).

APPENDIX

A3. Positions 121-175 correspond to fragment CB-IIIA and positions 176-214 correspond to fragment CB-IIIB. The se- quence from 121 to 155 was determined with CB-IIIA (L49). Peptides Ti (121-137) and T2 (138-175) were used to confirm this portion of the sequence. These peptides were obtained from L49 as follows. The light chain, with disulfide bonds intact, was cleaved with CNBr and applied to Sephadex G-100 in 6 M urea/l M acetic acid to exclude intact light chain and partial cleavage products. The major peak, consisting of a mixture of fragment 1-120 and fragment 121-175 disulfide-bonded to frag- ment 176-214 (see Fig. 3) was fractionated by ion-exchange chromatography on SP-Sephadex C-25 in 6 M urea/l M acetic acid with a 0-0. 5 M NaCl gradient. The peak corresponding to the two disulfide-bonded fragments was citraconylated, re- duced with dithiothreitol, and alkylated with ethyleneimine; the two constituent fragments were then separated by gel filtra- tion on Sephadex G-75 in 0.1 M NH4HCO3. Fragment 121-175 was digested with trypsin, and citraconyl groups were removed by incubation at pH 3.5 overnight. The two resulting peptides, Ti and T2, were separated by gel filtration on Sephadex G-25 in 1 M acetic acid. The sequence of Ti was determined from posi- tions 121-134 and that of T2, from 138-155. From positions 155-174 the sequence was obtained from a single sequenator run of fragment CB-IIIA (L8-47) that had been succinylated and cleaved with hydroxylamine. The sequence was clear from 155 to 167 except that Asp and Asn were not definitely distin- guished. After position 167 the overlap was severe and residues 172 and 173 were not clearly identified; the presence of Asx and Lys at these positions is in agreement with the amino acid com- position of CB-IIIA (Table 1) and is homologous with the A2 se- quence. Met-175 was assigned from the results of CNBr frag- mentation of L49. The sequence of positions 176-212 is based on two separate sequenator runs of fragment CB-IIIB (L49). The two COOH-terminal residues (C-L) were determined by car- boxypeptidase digestion of L49, L952, and L72 and analysis of the COOH-terminal tryptic peptides of L49, L72, and L'5 (4, 6).

A2. The sequence shown is that published for L315 (4), except for the following changes, based on a reexamination in this study of the sequence of the COOH-terminal CNBr fragment (CB-Ill) of L315 (positions 176-214). We find residues 184 and 185 to be Ser-Asp instead of Asp-Ser; residues 200 and 202 are Glu and Asp instead of Gln and Asn; and residue 191, previously desig- nated Asx, is Asn.

Al. The sequence shown is that reported for L'04 (5).

Note Added in Proof. The sequence of L49, a A3 chain, has also been determined from positions 155-176 on a purified fragment, beginning at 155, obtained by hydroxylamine cleavage of the fully reduced, car- boxymethylated chain. The sequence was identical to that shown in Fig. 5 except that residue 163 was identified as Asp, 166 as Asn, 171 as Asp, and 172 as Asn (173 was Lys, as shown). A comparable fragment of L315,

a A2 chain, had exactly the same sequence in this region except that 171 and 174 were Gly and Phe, as shown in Fig. 5 and reported (4). Thus, the sequences and alignment for A3 and A2 from 163 to 175 are:

A3: DTS N PTKE DN KYM A2: DTS N PTKE GN KFM

We thank Erlinda Capuno and Vivien Igras for skillful technical as- sistance and Robert Sauer for making sequence determination facilities available. This work was supported in part by National Institutes of Health Research Grants CA-15472 and Al-08054 and a Center Grant (CA-14051) from the National Cancer Institute.

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