Amino Acid Sequence of the Light Chain from a Mouse Myeloma Protein with Anti-Hapten Activity: Evidence for a Third Type of Light Chain

Abstract

A tentative amino acid sequence has been determined for the light chain of a mouse A-myeloma (MOPC-315) protein with anti-2,4-dinitrophenyl activity. An unusual amino acid sequence in the carboxyl-terminal 104 residues suggests that the chain represents a new class of immunoglobulin light chain. The "variable" segment of this chain (amino-terminal 110 residues) differs at only eight positions from the corresponding segment of the lambda chain of a mouse M-myeloma (MOPC-104) protein with anti-dextran activity.The recent finding of myeloma proteins with antibody activity (e.g., 1-3) and the development of procedures for the preparation of conventional antibodies of remarkably restricted heterogeneity (4, 5) provide opportunities to correlate the primary structure of immunoglobulins and their specific reactivities with particular haptens and antigens. Accordingly, we are studying the structure of protein 315, an A-myeloma protein that binds 2,4-dinitrophenyl (Dnp) ligands with high specificity (6). In this paper, we present a tentative amino acid sequence for the light chain (LI5) of this protein. An unusual sequence suggests that this chain represents a new type, resembling immunoglobulin light chains of the X type more than those of the K type.Protein 315 was isolated from sera of mice bearing tranisplants of MOPC-315, a plasma cell tumor*, whose origin is described below. Fully reduced carboxymethylated or aminoethylated light chains were prepared as described (40), and digested with trypsin, thermolysin, or pepsin. The peptides were purified by ion-exchange chromatography on Dowex resins (7) or SE-Sephadex (8), and their amino acid sequences were determined by Edman degradation (9). The successively removed amino-terminal residues were identified by: subtractive analysis, gas-liquid chromatography of CH3CNS amino acids (9), the dansyl procedure (10) G-100 in 5 M guanidine -HCl, followed by chromatography on CM-cellulose in 8 M urea (11). Antisera to LI'' were prepared by immunizing rabbits with the Fab fragment of protein 315 in complete Freund's adjuvant, and absorbing the sera with K-and X-containing A-myeloma proteins that were produced by plasma cell tumors MOPC-460 and S-176*.A complete set of peptides was not isolated from any one enzymatic digest, but a combination of peptides from trypsin and thermolysin digests accounted for the amino acid composition of the chain (Table 1), and provided the basis for the provisional sequence shown in Fig. 1. As few overlaps have been established thus far, the alignment of peptides was based largely on homology with the complete amino acid sequence of the mouse X light chain that is produced by plasma cell tumor MOPC-104 (12); this light chain is hereafter referred to as L104. There are two methionine residues in L315, and the three purified cyanogen bromide fragments obtained from this chain (corresponding to the N-terminal 87 residues, the central 88 residues, and the C-terminal 39

Lambda2 light chains in normal mouse immunoglobulins.

“…This suggests that VX2 and VX1 belong to separate V subgroups and are probably derived from different germline genes (18). This view is strengthened by recent evidence that there are two V genes in mouse embryonic DNA (19) and that one of them corresponds almost exactly to the V segment of L315 from the NH2-terminus to position 98; the only discrepancies are in two framework and three hypervariable region codons (20), and one of the framework differences (Gln for Glu at position 6) probably represents an error in the reported amino acid sequence (7,8).…”

“…The more prevalent A chains were designated.A1, and L315 was tentatively designated a A2 chain (7,8), implying that there are other L chains with the same constant domain.…”

“…About 95% in the mouse are throught to be K (2); of several hundred mouse myeloma proteins (3), only 21 with A chains have been identified (4). The existence of at least two types of L chain was suggested by the L chain made by mouse myeloma tumor MOPC-315 (5): this chain (LI'5) is A-like but differs from the other A chains at 29% of the COOH-terminal 110 positions ("constant" domain; 6,7,8). The more prevalent A chains were designated.A1, and L315 was tentatively designated a A2 chain (7,8), implying that there are other L chains with the same constant domain.…”

“…This article must therefore be hereby marked "advertisement" in accordance with 18 (4,6), and L315 (7,8) 18 hr at 370). The digest was freeze-dried and taken up in the first buffer of the peptide map (Fig.…”

“…The more prevalent A chains were designated.A1, and L315 was tentatively designated a A2 chain (7,8), implying that there are other L chains with the same constant domain.To search for A2 chains in normal mouse Igs we exploited the expectation that a tryptic digest of a mixture of K, Al, and X2 chains would yield the respective COOH-termini as lysine-and arginine-free peptides with the sequences shown in Fig. 1.…”