The amino acid sequence of rabbit J chain in secretory immunoglobulin A.

The primary structure of rabbit J chain, which occurs covalently bound to secretory IgA, was determined. J chain was isolated in its S-carboxymethylated form, in one step, by SDS/PAGE followed by electro-elution; 5 nmol of protein (approx. 75 micrograms), in all, was necessary for the determination of the complete sequence by the 'shot-gun' microsquencing technique; with the use of several site-specific endoproteinases, the various digests of S-carboxymethylated J chain were separated by micro-bore reverse-phase h.p.l.c. and the partial N-terminal sequences of all peptides were analysed. From the sequence alignment, gaps were filled by further extensive sequencing of the relevant overlapping fragments isolated from selected digests. Rabbit J chain comprises 136 amino acid residues, out of which eight are conserved cysteine residues, and is more closely similar to the human sequence (73.5% identify) than to the mouse sequence (68% identity). There is one unique glycosylation site at asparagine-48.     

Sequence studies on the heavy chain of rabbit immunoglobulin A of different alpha-locus allotypes.

The amino acid sequence was determined of part of the variable region of heavy chain from rabbit immunoglobulin A of allotypes a1 and a3. Two corrections of the primary sequence of Aa1 gamma-chains are reported; most of the structural correlates of the alpha-locus allotypes are confirmed. The amino acid sequence of the N-terminal 20 residues of alpha-negative molecules was also determined and found to be homologous to the human VhIII subgroup. These molecules are present in a much higher proportion in the alpha-chain pool than in the gamma-chain.     

Partial amino acid sequence of a rabbit immunoglobulin light chain of allotype b5

The amino acid sequence of a rabbit immunoglobulin light chain of allotype b5 has been nearly completed. A comparison of its structure with that of light chains of allotypes b4, b6, and b9 confirms that the constant regions of these various kappa chains differ by 20-35%. The substitutions are clustered in parts of the second half of the chain, and the b5 form bears more resemblance to the b6 chain than to any other, in good agreement with previous serological data. The analysis of the variable region reveals the existence of certain allotype-associated residues which have also been reported in other b5 chains, but not in proteins of the other allotypes. An examination of the rabbit light chain sequences between positions 96 and 107 suggests that this portion of the chain may be encoded separately by a joining "J" DNA segment, as has been described previously for murine and human immunoglobulins. In the rabbit, however, these J kappa regions appear to differ from one allotype to another. Together with the extensive variations of the constant regions, these data suggest that the rabbit kappa gene organization more closely resembles the murine gamma system (four different C gamma genes each flanked by its J segment) than the murine kappa system (a single C kappa gene).     

Allotype-related sequence variation of the heavy chain of rabbit immunoglobulin G

The heavy chain of rabbit immunoglobulin G exists in three major allotypic patterns, Aa1–Aa3. A comparison of the amino acid compositions of the heavy chains isolated from immunoglobulin IgG homozygous for each allotypic determinant revealed the presence of an additional methionine residue per chain in the Aa3 allotype relative to the Aa1 and Aa2 allotypes. The position of the additional methionine residue was determined by cyanogen bromide cleavage and by tryptic digestion of the γ-chains; it coincided with the inter-Fd–Fc area of the chain. Isolation and characterization of the corresponding tryptic peptides of 31 amino acid residues from each of the allotypes showed the presence of a methionine-for-threonine replacement in the Aa3 allotype, but only in about 70–80% of the molecules. No other allotypic variations were seen in this tryptic peptide. Allotypically related variations in composition were also detected in the N-terminal cyanogen bromide-cleavage peptide.     

The N-terminal sequence of the heavy chain of rabbit immunoglobulin IgG

The absence of an N-terminal amino acid with a free alpha-amino group from the heavy chain of rabbit immunoglobulin IgG has been confirmed and no evidence could be found of a blocking formyl, acetyl or propionyl group. The N-terminal amino acid appears to be pyrrolid-2-one-5-carboxylic acid (PCA) in all molecules. A mixed amino acid sequence follows in the approximate proportions: PCA-Ser-Val-Glu-Glu-Ser-Gly-Gly-Arg, 50%; PCA-Ser-Leu-Glu, 20%; PCA-Glu(NH(2)), 20%. The heavy chains of a purified antibody, namely anti-(human serum albumin), and of immunoglobulin IgG from a rabbit homozygous at the allotypic loci both showed a similar mixed N-terminal sequence.     

The structural basis of rabbit VH allotypes: serologic studies on a1 H chains with defined amino acid sequence.

The amino acid sequences for the VH regions of three homogeneous antibodies elicited by type III pneumococcal vaccine were determined. All three antibodies had the group a allotype a1. Two of the antibody H chains (3372, 3381) had identical amino acid sequences in all framework positions that are considered correlates of the VH allotype, whereas the third H chain (3T72) differed from these at positions 15 and 16. The a1 allotypic specificities of the three homogeneous antibodies were compared by quantitative radiobinding and inhibition assays by using both insolubilized anti-a1 antisera and allotypic antiserum fractions rendered specific for the homogeneous antibody 3374. It was found that antibodies 3374 and 3381 are allotypically indistinguishable and have in common an a1 allotypic specificity that predominates in pooled a1 IgG. The allotypic specificity of the 3T72 antibody, on the other hand, was markedly deficient to those of 3374, 3381, and the a1 IgG pool. This correlation of allotypic difference with amino acid sequence variation at position 15 and 16 of the H chain indicates the involvement of these two residues in a major a1 allotypic determinant.     

Partial amino acid sequence in the N-terminal region of an anti-pneumococcal immunoglobulin heavy chain of allotype a2 (Short Communication)

The amino acid sequence of the N-terminal 48 residues of the heavy chain derived from a homogeneous rabbit antibody to type III pneumococci is described. This chain of allotype a(2) is compared with other rabbit heavy chains of allotypes a(1), a(2) and a(3). Within the N-terminal 25 positions, two chains which carry the same allotype a(2) possess identical amino acid sequences, but differ markedly from heavy chains of allotypes a(1) and a(3). Sequence variability is observed in residues 26-27 and 30-34, but not in residues 35-48.     

Molecular analysis of recombination sites within the immunoglobulin heavy chain locus of the rabbit

Previously, recombinations involving genes of the rabbit immunoglobulin heavy chain locus have been documented serologically. These data indicated that the sites at which the causative recombination events occurred could have been anywhere from within the V _H gene cluster up to, or 3 of, C. Since these sites could not be localized further by serological methods, we attempted to do this using techniques of molecular biology. DNAs from homozygous recombinant rabbits and from the appropriate non-recombinant parental haplotypes were characterized using Southern blots hybridized with a panel of probes derived from cloned regions of the rabbit immunoglobulin heavy chain gene complex. In all three recombinants, the site was downstream of the entireV _H cluster and upstream of the J _Hcluster within an 50 kilobase (kb) egion containing expanses of repetitive-sequence DNA as well as D _H genes. D _H-specific probes further showed that in two of the recombinants, the recombination appears to have occurred within or 5 of D _H1 and 5 of D _H2 genes; in the third it occurred 3 of the D _H2 genes but at least 5 kb 5 of the J _H region. Address for correspondence and offprint request to: R. G. Mage.     

Amino acid sequence of rabbit skeletal muscle myosin. 50-kDa fragment of the heavy chain

The amino acid sequence of the 50-kDa fragment that is released by limited tryptic digestion of the head portion of rabbit skeletal muscle myosin was determined by analysis and alignment of sets of peptides generated by digestion of the fragment at arginine or methionine residues. This fragment contains residues 205-636 of the myosin heavy chain; among the residues of particular interest in this fragment are N epsilon-trimethyllysine, one of four methyl-amino acids in myosin, and Ser-324, which is photoaffinity labeled by an ATP analogue (Mahmood, R., Elzinga, M., and Yount, R. G. (1989) Biochemistry 28, 3989-3995). Combination of this sequence with those of the 23- and 20-kDa fragments yields an 809-residue sequence that constitutes most of the heavy chain of chymotryptic S-1 of this myosin.     

The N- and C-terminal amino acid sequences of the heavy chain from a pathological human immunoglobulin IgG

The heavy chain of a pathological human immunoglobulin IgG and also the Fd fragment have been isolated. No free alpha-amino group was present on either and the N-terminal sequence of both has been identified as pyrrolid-2-one-5-carbonylvalylthreonine. Splitting at the four methionine residues of the heavy chain with cyanogen bromide gave five fractions. The fraction from the C-terminal end of the chain was isolated in high yield and the amino acid sequence was: His-Glu-Ala-Leu-His-Asp(NH(2))-His-Tyr-Thr-Glu(NH(2))-Lys-Ser-Leu-Ser-Leu-Ser-Pro-Gly These results give strong support to the view that the heavy chain of immunoglobulin is a single peptide chain.