Primary structure of human J chain: alignment of peptides from chemical and enzymic hydrolyses

Abstract

The primary structure of the J chain from a human Waldenströms IgM protein has been determined using a combination of automated and conventional Edman degradative procedures. Eighty-five percent of the sequence was established with peptides isolated from tryptic digests of carboxyamidomethylated and citraconylated J chain, many of which were sequenced completely. Alignment of the tryptic fragments was achieved with peptides generated by chymotrypsin and limited acid hydrolyses. The j chain consits of 129 amino acids and a single oligosaccharide structure linked to asparagine at positon 43 of the sequence. The molecular weight, including 7.5% carbohydrate by weight, is 16 422. The location and arrangement of three half-cystines could be deduced from previous studies, whereas the pairing of the remaining five disulfide bonds still needs to be clarified.

Partial amino acid sequence of human factor D:homology with serine proteases.

“…Automated.Edman degradations were performed in the presence of 2 mg of Polybrene by using 0.5 M Quadrol and the program described by Brauer et al. (12,13). Prior to the sequence run, five degradation cycles were carried out without delivering heptafluorobutyric acid to remove glycine eluted from the gel slices.…”

Primary structure of the immunoglobulin J chain from the mouse.

“…Such studies have been hampered by the lack ofmutant forms of J chain that display altered function as well as by the difficulty in isolating enough J chain for detailed biochemical characterization. Because the J chain comprises a minor fraction of the polymer protein and is highly susceptible to enzymatic degradation (8), only the human J protein has been obtained in sufficient quantities to permit sequence determination (9). Finally, it has not been possible to study the native conformation ofJ chain as the sreducing conditions required to free the J chain from Ig polymers also reduce the intra-J chain disulfide bonds (10).…”

“…The murine J chain mRNA was found to encode at least 160 amino acids, more than that expected for the mature J protein. By comparing the mouse data with the amino acid sequence determined for the human J protein (9), the extra amino acids appeared to comprise part of an amino-terminal signal peptide. The precise boundary between the signal peptide and the mature J peptide could not be definitely assigned because the amino-terminal residue ofthe mouse J chain has yet to be identified.…”

“…3), it seems likely that the signal peptidase cleaves between a glycyl and an aspartyl residue to yield a signal peptide of at least 23 amino acids and a mature J chain of 137 amino acids. Cleavage at this point would mean that Jc21 cDNA contains most of the prepeptide information but lacks a few 5' codons including the initial AUG. Cleavage at this point would also imply that the mouse J chain has an unblocked amino-terminal residue in contrast to the cyclized glutamic acid found in the human J chain (9).…”

“…Although the proposed two-domain arrangement is substantiated by experimental data, many of the intradomain features-e.g., the assignment of intrachain bonds and the interaction of hydrophobic regionsremain purely hypothetical. It may be possible to ascertain the location ofthe J chain disulfide bridges by using chemical cleavage methods (9), but the resolution of other features of the J chain structure will require analysis of the three-dimensional structure ofthe parent polymers. Until such studies are accomplished, the proposed model can serve as a useful framework for probing the structure-function relationships of the J chain.…”