The Primary Structure of a Monoclonal Human λ‐Type Immunoglobulin L‐Chain of Subgroup II (Bence‐Jones Protein NEI)

Abstract

Protein NEI, which has a molecular weight of 23500 and contains 216 amino acids, was isolated from the urine of a patient with multiple myeloma by salt-precipitation and purified by ion-exchange chromatography, block electrophoresis and gel filtration. 23 tryptic peptides have been isolated from the aminoethylated protein and 13 chymotryptic peptides from the carboxymethylated protein by ion-exchange chromatography. Sequence studies have mainly been carried out with the tryptic peptides ; the chymotryptic peptides have been used for overlaps in the variable region, since the alignment of the tryptic peptides by homology was not unambiguous.Protein NEI belongs to subgroup I1 of the A-chains. It has a series of subgroup specific exchanges and a subgroup specific deletion a t position 96. Protein NEI differs from all other A-chains of subgroup I1 by possessing a sixth cysteine residue. This additional cysteine residue a t position 88 is so far unique, as it is adjacent to an invariant cysteine a t position 87. Protein NEI also contains a carbohydrate moiety attached to an aspartic acid or asparagine (Asx) residue a t position 93, one of the hypermutable areas of the variable part. Furthermore we conclude by comparison with other A-chains that all completely elucidated A-chains can be ordered into four subgroups. The subgroups and linkage groups within subgroups are in complete agreement with an evolutionary origin of antibody variability and are incompatible with somatic hypermutation models.Structural studies with monoclonal immunoglobulins have provided the first insight into the genetic mechanism of antibody formation [l--51. Since the molecule is divided into a variable and a constant part, the genetic control of the immunoglobulins is more complicated than with other protein families.There is, however, an increasing amount of evidence, that the extreme heterogeneity of the variable parts, which is responsible for the specificity of the antibody, is genetically determined. Crucial in this respect is the high order of regularity which governs the amino acid exchanges and deletions of the variable parts. These linked amino acid exchanges (subgroup and subsubgroups) can only be explained by an evolutionary process [3,6--81.One of the proteins which has contributed to the understanding of this variability is Bence-Jones protein NEI, a A-type L-chain of subgroup 11. The primary structure of this protein has been reported previously in a short communication

Three-Dimensional Structure of the Fab′ Fragment of a Human Immunoglobulin at 2.8-Å Resolution

“…However, contacts with antigens should be appreciably modified by this relatively small structural change. Similar considerations apply to positions 96 and 97, which are also considered to be subgroup-related deletions in human X chains (18), and to deletions (or additions) observed in H-chain sequences. Positions 86-91, which are hypervariable in human subgroup III H-chain sequences (19), are not close to the hypervariable sequences mentioned above and are not in contact with Lchain residues, although they occur at an outside region of the molecule (Fig.…”

Primary structure of a deleted human lambda type immunoglobulin light chain containing carbohydrate: protein Sm lambda.

“…Hydrazinolysis of the carboxypeptidase digest liberated serine, establishing the sequence of the NH2-terminal region as PCASer-Ala-Leu. This sequence is identical to that of the first four residues of the reference lambda chains of subgroup II, Nei and Bo (18,22).…”

“…The sequence determination of the peptides or the sub-peptides derived therefrom by further enzymatic cleavage was performed by the Edman-dansyl technique (20) or occasionally by subtractive Edman degradation (21). The COOHterminal residue of the peptides was established or confirmed by amino-acid analysis following treatment with carboxypeptidase A and/or B (18). The side-chain charge of glutamic acid or glutamine and of aspartic acid or aspara- procedure (18), serine was shown to be the COOH-terminal residue.…”

“…The COOHterminal residue of the peptides was established or confirmed by amino-acid analysis following treatment with carboxypeptidase A and/or B (18). The side-chain charge of glutamic acid or glutamine and of aspartic acid or aspara- procedure (18), serine was shown to be the COOH-terminal residue. Thus, both NH2-and COOH-terminal residues were identical to those of other normal lambda chains.…”

“…After purification of the protein by gel filtration on Sephadex G-100 in 1 N propionic acid, the amino-acid sequence was defined by isolation and purification of the tryptic and chymotryptic peptides from the aminoethylated or carboxymethylated protein, respectively, according to the procedures outlined by Garver and Hilschmann (18). Following chymotryptic digestion of the alkylated protein, the NH2-terminal peptide was selectively isolated from a column of Dowex 50 X2 equilibrated with water by the method of Press et al (19).…”