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.     

Amino acid sequence of the N-terminal sixty-nine residues of heavy chain derived from a homogeneous rabbit antibody

The sequence of the N-terminal 69 residues of heavy chain from a homogeneous rabbit antibody to type III pneumococcal polysaccharide was determined. The sequence is similar to that found in heavy chains of normal pooled rabbit immunoglobulins of the same allotype Aa1. Two regions of the homogeneous heavy chain (residues 35-46 and 62-69) are very similar to corresponding regions of heavy chains from rabbit Aa2 immunoglobulin, as well as from mouse, guinea-pig and human immunoglobulins. In contrast, residues 47-62 appear to be variable. Comparison in this section with another homogeneous anti-pneumococcal antibody (Strosberg et al., 1972) of related specificity and of the same allotype indicates sequence variation in at least three positions. An antibody to group C streptococcal carbohydrate of allotype Aa2 (Fleischman, 1971) differs by five amino acids in the same region of the heavy chain. Sequence variability between these three antibodies does not occur in homologous positions within this variable section. Allotype-related sequences could not be identified in section 34-65.     

The partial sequence of two large peptides from the N-terminal half of heavy chains from normal rabbit immunoglobulin G

The partial amino acid sequence of two large peptides is described. These were prepared from the N-terminal half of the heavy chain of immunoglobulin G from pooled normal rabbit serum by tryptic digestion after the in-amino groups of the lysine residues had been blocked with S-ethyl trifluorothioacetate. These peptides are believed to account for about 145 residues of fragment C-1, the N-terminal section of rabbit immunoglobulin G heavy chain prepared by cyanogen bromide cleavage. The evidence from the present paper and the preceding paper (Cebra, Givol & Porter, 1968) suggests that it may be possible to deduce a predominant amino acid sequence for most, if not all, of this section of the molecule.     

Allotypically related sequences in the Fd fragment of rabbit immunoglobulin heavy chains

The sequence has been completed of the N-terminal 94 residues of the variable section of the Fd fragment of heavy chains from rabbit immunoglobulin G (IgG) of allotype As1. Most of the sequence of the same section from IgG of allotype Aa3 is also reported. These results, in conjunction with a substantial sequence of the variable region of allotype Aa2 reported elsewhere (Fleischman, 1971), show the presence of 16 positions (including six consecutive positions) in which the residue present correlates with the allotype. No allotype-related sequence variation has been found in the constant section of the Fd fragment. This evidence supports the view that two genes code for the heavy chain and it can be used as evidence in favour of somatic mutation as the origin of the variability in the sequence of the N-terminal section. The evolutionary origin of the ;a' locus allotypes of rabbit immunoglobulins remains obscure.     

Amino acid sequences of the human kidney cathepsins H and L

The complete amino acid sequences of human kidney cathepsin H (EC 3.4.22.16) and human kidney cathepsin L (EC 3.4.22.15) were determined. Cathepsin H contains 230 residues and has an Mr of 25116. The sequence was obtained by sequencing the light, heavy and mini chain and the peptides produced by cyanogen bromide cleavage of the single-chain form of the enzyme. The glycosylated mini chain is a proteolytic fragment of the propeptide of cathepsin H. Human cathepsin L has 217 amino acid residues and an Mr of 23720. Its amino acid sequence was deduced from N-terminal sequences of the heavy and light chains and from the sequences of cyanogen bromide fragments of the heavy chain. The fragments were aligned by comparison with known sequences of cathepsins H and L from other species. Cathepsins H and L exhibit a high degree of sequence homology to cathepsin B (EC 3.4.22.1) and other cysteine proteinases of the papain superfamily.     

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.     

A base substitution at a splice site in the COL3A1 gene causes exon skipping and generates abnormal type III procollagen in a patient with Ehlers-Danlos syndrome type IV

The dermis of a child with Ehlers-Danlos syndrome type IV (EDS-IV) contained about 11% of the normal amount of type III collagen and cultured dermal fibroblasts produced a reduced amount of type III procollagen which was secreted poorly. Type III collagen produced by these cells contained normal and abnormal alpha-chains and cyanogen bromide peptides. The site of the structural defect in the abnormal alpha 1 (III) chains was localized to the region of Met797, which is at the junction of the two carboxyl-terminal CB5 and CB9 cyanogen bromide peptides. Chemical cleavage of heteroduplexes formed between EDS-IV mRNA and a normal cDNA clone covering the CB5 and CB9 region showed that about 100 nucleotides were mismatched. Sequencing of amplified and cloned cDNA spanning the mutant region revealed a 108 nucleotide deletion corresponding to amino acid residues Gly775 to Lys810. The deleted nucleotide sequence corresponded to sequences that, by analogy to the organization of the type I collagen genes, should be precisely encoded by exon 41 of the COL3A1 gene. Sequencing of amplified genomic DNA, prepared using disimilar amounts of primers specific for exons 41 and 42, displayed a base substitution (G-to-A) in the highly conserved GT dinucleotide of the 5' splice site of intron 41. Normal sequences were also obtained from the normal allele. It is likely that the GT-to-AT transition at the splice donor site of intron 41 generated an abnormally spliced mRNA in which sequences of exon 40 and 42 were joined together with maintenance of the reading frame. The corresponding peptide deletion included the cyanogen bromide cleavage site Met797-Pro798 and the mammalian collagenase cleavage site at Gly781-Ile782. These losses account for the resistance of EDS-IV collagen to cyanogen bromide and mammalian collagenase digestion. Cultured fibroblasts produced normal homotrimer, mutant homotrimer, and mixed heterotrimer type III collagen molecules. The mutant homotrimer molecules were the major pepsin-resistant species and about 69% of the alpha 1(III) mRNA was in the mutant form.     

The isolation and characterization of the V-H domain from rabbit heavy chains of different a locus allotype.

The Fd fragment of rabbit gamma-chains was split by papain to yield a smaller fragment with a molecular weight of approximately 14,000 and dialyzable small peptides and amino acids. The domain size fragment was identified as intact variable region from its amino acid content, its blocked amino-terminus, and two characteristic cysteine-containing peptides, while the small peptides and amino acids were accounted for by the degradation of the C-H1 region. The variable regions isolated from Aa1 and Aa3 Fd fragments not only reacted quantitatively with immunoadsorbents conjugated with the homologous anti-a allotype antibody, but also completely inhibited the binding of the parent Fd fragment to the homologous antibody as measured by radioimmune assay. These data provide direct evidence that the group a allotypic determinants are contained entirely in the variable portion and are independent of the constant portion of rabbit heavy chains.     

Comparison of the amino acid sequences of the variable domains of two homogeneous rabbit antibodies to type III pneumococcal polysaccharide.

The amino acid sequences of the V (variable) regions of the H (heavy) and L (light) chains derived from rabbit antibody K-25, specific for type III pneumococci, were determined; this is the second homogeneous rabbit antibody besides antibody BS-5 whose complete sequence of the V domain has been established (Jaton, 1974d). The V regions of L chains BS-5 and K-25 (both of allotype b4) differ from each other by 19 amino acid residues; 11 of these 19 substitutions are located within the three hypervariable sections of the V region. On the basis of seven amino acid differences within the N-terminal 28 positions, it is suggested that L chain K-25 belongs to a different subgroup of rabbit K chains and L chain BS-5. H chain K-25 (allotype a2) differs from another H chain of the same allotype by one amino acid substitution within the N-terminal 70 positions in addition to interchanges occurring in the first two hypervariable sections. H chain K-25 was compared with H chain BS-5 (allotype a1) and with the known V-region rabbit sequences. Allotype-related differences between a1, a2 and a3 chains appear to occur within the N-terminal 16 positions and possibly in scattered positions throughout the V-region. In the hypervariable positions, variability between the two antibodies is remarkably more pronounced within the third hypervariable section of both H and L chains than within the first two.     

Hemocyanins in spiders, VI[1]. Comparison of the polypeptide chains of Eurypelma californicum hemocyanin.

The subunits of the hemocyanin from the tarantula, Eurypelma californicum, were isolated, following dissociation at pH 9.6, by a sequence of chromatographic and electrophoretic steps. Fraction 2 (containing two chains, a and c2) and the constituent polypeptide chains of the dimeric subunit 4D (b and c4) were resolved by anion exchange chromatography at pH 8.9 and 6.5, respectively. Since c2 and c4 have different electrophoretic mobilities in polyacrylamide gradient gels, the total number of different polypeptide chains is seven. The amino acid compositions of the seven chains are reported. There are major differences for at least half of the amino acids, while more consistent proportions become evident, if the amino acids are grouped by types of side chains. The N-terminal amino acid is proline in the case of chains b and e,, while no end group called be detected in any of the other chains by different methods. The C-terminal end group was found to be valine in both chains d and e. Cleavage by 70% formic acid, and by cyanogen bromide in formic acid results in fragmentation patterns distinct for each chain. After cyanogen bromide cleavage, the two largest peptides of each chain are of molecular weight near 2400. Tryptic fingerprints also reveal significant differences between all chains. Subunit heterogeneity of Eurypelma hemocyanin is clearly not the consequence of secondary modifications, but resides in major differences of the amino acid sequences.     

Amino acid sequence of rat liver cathepsin L

The complete amino acid sequences of the heavy and light chains of rat liver cathepsin L (EC 3.4.22.15) were determined at the protein level. The heavy and light chains consisted of 175 and 44 amino acid residues, respectively, and their Mr values without glycosyl groups calculated from these sequences were 18941 and 5056, respectively. The amino acid sequence was also determined from the N-terminal sequences of the heavy and light chains, and the sequences of cleavage fragments of the heavy chain with lysylendopeptidase and cyanogen bromide. The fragments were aligned by comparison with the amino acid sequence deduced from the sequence of cDNA of rat preprocathepsin L. The sequence of rat liver cathepsin L determined at the protein level was identical with that deduced from the cDNA sequence except that in the heavy chain, residues 176-177 (Asp-Ser) were not present at the C-terminus and alanine was replaced by proline at residue 125. Asn-108 in the heavy chain is modified with carbohydrate.     

Outer membrane proteins of Escherichia coli. V. Evidence that protein 1 and bacteriophage-directed protein 2 are different polypeptides.

Protein 1 from the outer membrane of Escherichia coli K-12 and protein 2 from a phage PA-2 lysogen of the same strain were isolated by differential sodium dodecyl sulfate extraction and purified by ion-exchange and gel filtration chromatography. Rabbit antisera were prepared against these proteins and showed no cross-reaction between proteins 1 and 2. The proteins have the same N-terminal amino acid but show small yet significant differences in amino acid composition. The proteins were cleaved with cyanogenbromide in solvents containing both formic acid and trifluoroacetic acid. By comparing the cleavage in these solvents, it was established that protein 1 yielded 5 cyanogen bromide peptides, and the sum of the molecular weights of these was equivalent to the molecular weight of the uncleaved protein. Protein 2 yielded 4 cyanogen bromide peptides, none of which was identical to those of protein 1, and the sum of these peptides was also equivalent to the apparent molecular weight of the uncleaved protein. Significant differences were also observed when tryptic peptides from the two proteins were compared. These results indicate that protein 1 and the phage-directed protein 2 are distinct, different, and apparently homogeneous proteins.     

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.     

Antigenic determinants in the disulfide regions of bovine fibrinogen.

Rabbit antibodies to bovine fibrinogen were used to study the antigenic activity of four cyanogen bromide peptides containing the disulfide regions of this molecule. In precipitation tests the highest activity was associated with the peptide F-CB3 which is exclusively derived from the alpha-chain. Reduction of the single disulfide bridge in peptide F-CB3 did not influence its serologic activity. Weaker reactions were observed with the N-terminal multichain peptide F-CB1. The antigenicity of peptide F-CB1 was not affected by removal of fibrinopeptides A and B but it was lost after reduction. The immunological activity of the multichain peptide F-CB2 was even less than that of peptide F-CB1 and the antigenic determinants were destroyed by reduction. A large fragment essentially composed of peptides F-CB1 and F-CB2 could be obtained by limited cyanogen bromide cleavage and showed considerably better immunological activity than peptides F-CB1 and F-CB2 together. Apparently no activity was associated with a mixture of small hydrophobic, disulfide-loop peptides tentatively called peptide F-CB4. The large loss of antigenic activity in cyanogen bromide digests of fibrinogen suggests that the disulfide-stabilized regions do not have an important role in maintaining conformational antigenic determinants of fibroinogen. Changes in noncovalently stabilized conformation requiring uncleaved chains is considered as a possible reason for the findings observed.     

Sequence studies on the constant region of the Fd sections of rabbit immunoglobulin G of different allotype.

The amino acid sequence has been completed for the constant region of the Fd fragments of heavy chains from rabbit IgG (immunoglobulin G) of allotype Aa1 and Aa3. The amino acid sequence given by Fruchter et al. [(1970) Biochem. J. 116, 249-259] for the constant region of the Fd fragment from Aa1 IgG was extended and in in part corrected to give a continuous sequence of 140 residues. No allotype-related sequence variation was found in the constant section of the Fd fragment. This evidence confirms the view that the differences in sequence between the variable regions of Aa1 and Aa3 IgG [Mole et al., (1971) Biochem. J. 124, 301-318] are responsible for the allotypic specificities.     

Amino acid sequences around the cysteine residues of rabbit muscle triose phosphate isomerase

1. The nature of the subunits in rabbit muscle triose phosphate isomerase has been investigated. 2. Amino acid analyses show that there are five cysteine residues and two methionine residues/subunit. 3. The amino acid sequences around the cysteine residues have been determined; these account for about 75 residues. 4. Cleavage at the methionine residues with cyanogen bromide gave three fragments. 5. These results show that the subunits correspond to polypeptide chains, containing about 230 amino acid residues. The chains in triose phosphate isomerase seem to be shorter than those of other glycolytic enzymes.     

Primary structure of the OSCP protein that confers sensitivity to oligomycin on the mitochondrial H+-ATPase complex. I. Tryptic and cyanogen bromide peptides

Trypsin and cyanogen bromide were used for cleavage of the OSCP preparations. The peptide mixtures thus formed were separated into individual components by a combination of various chromatographic procedures: gel filtration, ion exchange and paper chromatography, as well as reversed-phase HPLC. As a result, 31 tryptic peptides and 9 out of 10 possible cyanogen bromide peptides were isolated. Determination of the amino acid sequences of these peptide allowed the alignment of cyanogen bromide fragments in the polypeptide chain that shed light on the "architecture" of the protein molecule as a whole. It also afforded the overlappings for tryptic peptides, 16 in the N-terminal and 8 in the C-terminal portions of the molecule.     

Major carbohydrate structures at five glycosylation sites on murine IgM determined by high resolution 1H-NMR spectroscopy

Mouse myeloma immunoglobulin IgM heavy chains were cleaved with cyanogen bromide into nine peptide fragments, four of which contain asparagine-linked glycosylation. Three glycopeptides contain a single site, including Asn 171, 402, and 563 in the intact heavy chain. Another glycopeptide contains two sites at Asn 332 and 364. The carbohydrate containing fragments were treated with Pronase and fractionated by elution through Bio-Gel P-6. The major glycopeptides from each site were analyzed by 500 MHz 1H-NMR and the carbohydrate compositions determined by gas-liquid chromatography. The oligosaccharide located at Asn 171 is a biantennary complex and is highly sialylated. The amount of sialic acid varies, and some oligosaccharides contain alpha 1,3-galactose linked to the terminal beta 1,4-galactose. The oligosaccharides at Asn 332, Asn 364, an Asn 402 are all triantennary and are nearly completely sialylated on two branches and partially sialylated on the triantennary branch linked beta 1,4 to the core mannose. The latter is sialylated about 40% of the time for all three glycosylation sites. The major oligosaccharide located at Asn 563 is of the high mannose type. The 1H-NMR determination of structures at Asn 563 suggests that the high mannose oligosaccharide contains only three mannose residues.     

Separation of cyanogen bromide-cleaved peptides of the vesicular stomatitis virus glycoprotein and analysis of their carbohydrate content.

The purified glycoprotein of vesicular stomatitis virus was cleaved at methionine residues with cyanogen bromide, and the resultant peptides were analyzed by two-dimensional electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. Five peptide bands were resolved in cylindrical gels run under nonreducing conditions. After reduction and electrophoresis in the second dimension, 11 peptides were resolved, indicating that several were originally linked by disulfide bonds. Double-label experiments indicated that at least 8 of the 11 peptides were unique. The major oligosaccharide chains were attached to two different cyanogen bromide peptides. In addition, six other peptides contained small amounts of sialic acid, fucose, and mannose, indicating that the glycoprotein contains more carbohydrate chains than the two major ones which have been reported previously.