Amino acid sequence of the N-terminal 139 residues of light chain derived from a homogeneous rabbit antibody

The amino acid sequence of the N-terminal 139 residues of the L (light) chain derived from a homogeneous rabbit antibody (designated BS-1) to type III pneumococci was determined. A combination of methods involving tryptic cleavage restricted to the 2 arginine residues of the molecule and mild acid hydrolysis of a labile peptide bond between the V (variable) and C (constant) regions of the L chain (Fraser et al., 1972) allowed the isolation of two large peptides comprising the entire V region (residues 1-109); these peptides were suitable for automated Edman degradation. The complete sequence analysis of the V region was carried out with only 4mumol of L chain. This material was homogeneous, although minor variant sequences, if present at the 10% value, would not have been detected. The L chain contains 3 intrachain disulphide bridges, whose pairing was established by diagonal electrophoresis: there is one V-region bridge between positions 23 and 88 and one C-region bridge between positions 134 and 194; the third one connects V and C domains between positions 80 and 171. When compared with the basic sequence of human kappa chains, rabbit L chain BS-1 appears to be more similar to the V(KI) prototype sequence than to V(KII) or V(KIII) sequences, where V(KI), V(KII) and V(KIII) represent subgroups I, II and III respectively of V regions of kappa light chains. The V regions of rabbit heavy and light chains are homologous to each other. The presence of two clusters of 3 glycine residues in positions 94-96 and 99-101 respectively is remarkable. Residues 94-96 may be related to antibody complementarity whereas residues 99-101 function probably as a pivot permitting the combining region of the L chain to make optimal contact with the antigenic determinant (Wu & Kabat, 1970).     

Amino acid sequence of the light chain derived from a rabbit anti-p-azobenzoate antibody of restricted heterogeneity

The amino acid sequence of the light chain from a specifically purified rabbit (No. 2717) anti-p-azobenzoate antibody preparation (b4 allotype) of restricted heterogeneity has been determined. This light chain is composed of 216 residues, including seven half-cystine residues located at positions 23, 80, 88, 134, 171, 194 and 216. Three intrachain disulfide bonds appear to be present in contrast to only two disulfide bonds as has been so far described for Bence Jones protein and light chains of human and mouse. This light chain was sequenced by isolating the tryptic peptides, sequencing the peptides and establishing their order within the molecule. Unambiguous identification of the overlaps was achieved by taking into account the partially characterized tryptic peptides from citraconic anhydride-treated light chains and chymotryptic and peptic peptides from digests of both untreated and citraconylated light chains. Comparison of this amino acid sequence with the amino acid sequence of the car?ylterminal half of the b4 light chains from unimmunized rabbits reveals differences at positions 165, 166, 169 and 176 indicating the existence of more than one sequence in the b4 “constant” region. There is substantial sequence homology between the variable half of 2717 light chain and human Bence Jones protein. Indeed, 46 positions in the V region (42%) are occupied by the same residues in this light chain and in human subgroup V_κIII.     

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.     

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.     

Amino acid sequence of the light chain variable region from a mouse anti-digoxin hybridoma antibody

A hybridoma cell line (26-10) derived from the A/J strain of mice secretes an immunoglobulin (IgG2a-k) which binds digoxin with an association constant of 1.2 nM. Such high-affinity antibodies have been utilized in clinical radioimmunoassays as well as in the reversal of toxicity due to excess digoxin. The amino acid sequence of the light chain variable region of this antibody was derived by automated sequencing of the following: the intact chain; a fragment beginning C terminal to the tryptophan residue 40, obtained by cleavage with iodosobenzoic acid; a fragment beginning C terminal to arginine residue 82, obtained by trypsin cleavage on the completely reduced, alkylated, and succinylated chain. Difficulties which had previously prevented the automated Edman sequencing of this chain (and, presumably, similar ones of the same subgroup) were overcome by increasing the duration of the cleavage step at proline residues 8 and 12. The sequences of the first two hypervariable and framework regions of this chain are virtually identical with those of the dinitrophenol- and menadione-binding myeloma light chain MOPC 460 (95% homology). This anti-digoxin hybridoma from the A/J strain makes use of a Vk gene which is similar to that utilized by some BALB/c 2,4-dinitrophenol-binding myelomas.     

Common peptides from the N-terminal half of heavy chain of immunoglobulin G from normal rabbit serum and a specific antibody

Fragment C-1, the N-terminal half of the heavy chain of rabbit immunoglobulin G, was prepared by cyanogen bromide cleavage from the heavy chain of immunoglobulin G obtained both from the pooled serum of normal rabbits and from specific anti-dinitrophenyl antibody. Tryptic digestion of fragment C-1 after the lysine residues had been allowed to react with S-ethyl trifluorothioacetate led to the isolation of six peptides from inert immunoglobulin G and specific antibody that appear to account for most of this section of the heavy chain. This approach should make possible comparative sequence studies of the Fd section of the heavy chain from different allotypes and from specific antibodies.     

Amino acid sequence of rabbit skeletal muscle myosin light chain kinase

The amino acid sequence of the amino-terminal, 235-residue segment of rabbit skeletal muscle myosin light chain kinase has been determined. Together with the carboxyl-terminal segment previously described [Takio, K., Blumenthal, D. K., Edelman, A. M., Walsh, K. A., Krebs, E. G., & Titani, K. (1985) Biochemistry 24, 6028], the present work completes the 603-residue sequence of this protein. The amino-terminal segment that has been analyzed herein corresponds to a domain reported to be of highly asymmetrical shape and as yet unknown function. Secondary structure calculations failed to provide any evidence of alpha-helix or beta-structures, but polyproline II like helical structure is possible. Sequence analysis indicates the presence of approximately equal quantities of two isoforms differing in a single amino acid replacement. Unexpected difficulties were encountered in the present sequence analysis due to the presence of acid-labile Asp-Pro bonds and to five separable variants of a blocked 21-residue amino-terminal peptide, arising from rearrangement at an Asn-Gly bond.     

Isolation of an active variable-domain fragment from a homogeneous rabbit antibody heavy chain. Physiochemical and immunological properties.

A fragment corresponding to most of the variable domain of the rabbit heavy chain (VH) was obtained by tryptic digestion of the midly reduced and aminoethylated heavy chain from rabbit antibody 3T72. The domain size peptide was purified by gel filtration and shown to extend between residues 11(Leu) and 122(Lys) of the heavy chain by sequence analysis. The molecular size of the fragment (approximately 11 000) was determined by gel filtration under denaturing conditions. Under nondenaturing conditions (20 mM sodium acetate, pH 5.5, 0.1 M NaCl), however, the fragment exists as a mixture of monomeric and dimeric species. The varable-domain fragment retains the allotypic determinants of the heavy chain (a1), as shown by double diffusion on agar plates and radioimmunoassay. Upon recombination of the heavy-chain variable-domain fragment with its homologous light chain, partial recovery of specific binding activity toward the SIII polysaccharide antigen was demonstrated. The method reported here is reproducible (with yields varying between 40 and 60%) and may provide a general method for obtaining the variable region of the heavy chain for antigen binding and allotypic and amino acid sequence studies.     

Nucleotide sequence of a rabbit IgG heavy chain from the recombinant F-I haplotype

We report the sequence of a cDNA encoding a rabbit immunoglobulin gamma heavy chain of d12 and e14 allotypes with high homology to partial cDNA sequences from rabbits of d11 and e15 allotypes. The encoded rabbit protein shows homologies with human (68-70%) and mouse (60-63%) gamma chains. The nucleotide sequence homologies of the CH domains range from 76-84% with human and 64-76% with mouse sequences. Comparison of the portion of VH encoding amino acid positions 34-112 with a previously determined VH sequence of the same allotype shows high conservation of sequences in the second and third framework segments but more marked differences both in length and encoded amino acids of the second and third complementarity-determining regions (CDRs). We also found a high degree of homology with a human genomic V-region, VH26 (77%) and a remarkable similarity between rabbit and human second CDR sequences and human genomic D minigenes. These results provide additional evidence that D minigene sequences share information with the CDR2 portion of VH regions.     

Amino acid sequence of acylphosphatase from rabbit skeletal muscle

The complete amino acid sequence of acylphosphatase from rabbit skeletal muscle has been elucidated by automatic Edman degradation of peptides obtained from staphylococcal protease and trypsin digestions. The enzyme consisted of a single polypeptide chain of 98 amino acid residues, lacking only histidine. Its amino (N)-terminus was blocked by an acetyl group. The presented sequence of rabbit muscle enzyme was compared with those of equine and porcine muscle enzymes. There were four unique replacements, i.e., Arg-4, Asp-28, Arg-31, and Glu-56 in the sequences of both equine and porcine muscle enzymes were replaced by Gly, Gly, Lys, and Asp, respectively, in that of rabbit muscle enzyme. Extensive structural homology was observed among the three enzymes.     

Amino acid sequence of the N-terminal 108 amino acid residues of the B chain of subcomponent C1q of the first component of human complement.

The amino acid sequence of the N-terminal 108 residues of the B chain of subcomponent C1q of the first component of human complement was determined. The B chain has a blocked N-terminal amino acid, which was judged to be 5-oxopyrrolidine-2-carboxylic acid. A collagen-like region of 84 residues was found, which started at position B-6, and all of the six hydroxylysine residues and 12 hydroxyproline residues present in the chain were found in this region. Four of the six hydroxylysine residues may be glycosylated. The repeating nature of the collagen-like region is broken at position B-9, where alanine is found in a position where glycine would be expected. The exact position of the interchain disulphide bond joining the A and B chains of human subcomponent C1q was shown to be between residues A4 and B4.     

Localization within the heavy chain sequence of tyrosyl peptides from the combining sites in a rabbit anti-3-azopyridine antibody preparation

A relatively homogeneous rabbit heavy chain was cleaved by CNBr. Fragment C-1 (the N-terminal half of the heavy chain) was isolated. Reduction and alkylation of C-1 liberated three fragments and partial sequence analysis of these isolated fragments showed that C-1 had been split on the carboxyl-side of Met 84. Similar results were obtained with another anti-hapten antibody preparation in which tyrosyl residues in the combining sites had been labeled. The labeled tyrosyl residues were found in the fragment representing residues 85–253. Since the constant region begins at about residue 120 and the sequences of the tyrosyl peptides from the combining sites are not present in published constant region sequences, these peptides appear to be derived from a variable region between residues 85 and 120.     

The V-region sequence of the H chain from a third rabbit anti-pneumococcal antibody.

The amino acid sequence of the V (variable) region of the heavy (H) chain of rabbit antibody BS-1, raised against type III pneumococcal vaccine, is reported. Together with the sequence data of the V region of the light (L) chain previously determined [Jaton (1974a) Biochem. J. 141, 1-13], the present work completes the analysis of the V domain of the homogeneous antibody BS-1. The V domains (VL + VH regions) of this antibody are compared with those of two other anti-(type III) pneumococcal antibodies BS-5 and K-25 [Jaton (1975) Biochem. J. 147, 235-247]. Except for the second hypervariable section of the L chains, these antibodies have very different sequences in the hypervariable segments of the V domains. Within the third hypervariable region of the H chain, each antibody has a different length: BS-1 is three amino acids shorter than K-25 and two amino acids shorter than BS-5. When the sequences in that section are aligned for maximal homology, only two residues, glycine-97 and leucine-101, are common to the three antibodies. On the basis of the amino acid sequences of these three anti-pneumococcal antibodies, the results do not support the concept of a simple correlation between primary structure in the hypervariable sections (known to determine the shape of the combining site) and antigen-binding specificity.     

Amino acid sequence of rabbit apolipoprotein E

The complete amino acid sequence of rabbit apolipoprotein E (apoE) was determined by generating three sets of peptides using cyanogen bromide, endoproteinase AspN, and Staphylococcus aureus V8 protease to cleave the protein. Through twenty cycles of sequence analysis on the whole protein, glutamic acid was identified as the N-terminal residue of rabbit apoE; the C-terminus of the protein was identified as glutamine. Based on the sequence of 294 amino acid residues determined by protein structure analysis, the molecular weight of rabbit apoE was determined to be 33,684. The protein sequence differed from the cDNA inferred sequence in 19 positions, only one of which could be attributed to microheterogeneity. The corrected amino acid sequence of rabbit apoE shares 80% homology with the human apoE sequence, 4% greater homology than that inferred from the cDNA sequence. The great similarity in the amino acid sequences of human and rabbit apoE suggests that their physical and physiological properties may also be similar. This homology and the relative ease with which apoE is isolated from rabbit plasma make it possible to conduct some in vitro experiments with the rabbit apoprotein that would have direct relevance to human apoE, but would be difficult or impossible with the human counterpart because of the quantity of protein required.