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.     

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.     

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.     

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 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.     

Partial amino acid sequence and genetic control of latent a2 allotype induced in rabbits by immunization with anti-a2 antibody

We have shown that after immunization of homozygous a1 rabbits of the B immunoglobulin (Ig) heavy chain haplotype with anti-a2 antibody (Ab) a population of molecules appears that has all of the serologic characteristics of the a2 allotype. We have now isolated these putative latent a2 molecules, have separated the heavy chains, and after enzymatic deblocking, have determined the first 19 N-terminal amino acids. For all eight allotype-associated residues, these putative latent a2 molecules have the amino acid residues typical of a2 allotype. As expected, the preimmune IgG from this a1a1 rabbit has the amino acids typical of the a1 allotype. Thus by partial amino acid sequence analysis, we provide additional evidence that the latent a2 allotype can be induced in a1a1 rabbits of the B heavy chain haplotype by immunization with anti-a2 Ab. Rabbits of other heavy chain haplotypes were also immunized with anti-a2 Ab and were tested for their ability to synthesize latent a2 allotype. Thus far, a1a1 rabbits of the A, B, C, and I heavy chain haplotypes all synthesize latent a2 allotype. In contrast, a3a3 rabbits of the G and H heavy chain haplotypes did not synthesize latent a2 allotype.     

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.     

Goat immunoglobin G. Peptide chains and terminal residues

Goat immunoglobulin G (IgG) was isolated and characterized. The molecular weights of the IgG and its heavy chains and light chains were found to be 144000, 53600 and 23000 respectively. The light chain corresponds to human L type as was shown by the absence of C-terminal S-carboxymethylcysteine and its high content of N-terminal pyrrolid-2-one-5-carboxylic acid (PCA). The major C-terminal residue of the light chain was serine and the major N-terminal dipeptide was PCA-Ala (0.6mole/mole). The major C-terminal residue of the heavy chain was glycine and the N-terminal sequence of the heavy chain is PCA-Val-Gln. This tripeptide was obtained in a 70% yield.     

Variation in the N-terminal sequence of heavy chains of immunoglobulin G from rabbits of different allotype

The sequences of the N-terminal peptides prepared by Pronase digestion of the heavy chain of rabbit immunoglobulin G of allotype Aa1, Aa2 and Aa3 were determined and were shown to be related to the allotype. An N-terminal fragment of about 34 residues was also prepared from the allotype heavy chains, by cleavage with cyanogen bromide; the yield varied with the allotype. The sequences of the cyanogen bromide fragments from the Aa1 and Aa3 heavy chains contain allotype-related variations similar to those found in the N-terminal Pronase peptides, and these sequences are thought to be representative of the whole heavy-chain populations. There is about 60% homology between the two sequences, and superimposed on the differences between them there are a number of positions within each sequence at which at least two amino acids are present.     

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.     

The formation of pyrrolid-2-one-5-carboxylic acid at the N-terminus of immunoglobulin G heavy chain

We propose that pyrrolid-2-one-5-carboxyl-tRNA is not involved in the initiation of protein synthesis in eukaryotic cells and that the N-terminal pyrrolid-2-one-5-carboxylic acid group of an IgG (immunoglobulin G) (that secreted by the mouse plasmacytoma Adj PC5) is formed by the enzymic cyclization of the N-terminal glutamine of the heavy chain of the completed IgG molecule and that the cyclization takes place inside the cell. We base these conclusions on the following evidence. (1) Pyrrolidonecarboxyl-tRNA was not found in incorporation experiments with rat liver preparations and [U-(14)C]-pyrrolidonecarboxylic acid, glutamic acid and glutamine, even though an incorporation extent of less than 2% of the total products could have been detected. (2) Double-labelling experiments showed that less than 8% of the nascent peptides of heavy chains (those obtained by precipitation by the antibody to Fc fragment) began with pyrrolidonecarboxylic acid. (3) Further double-labelling experiments showed that 60-66% of the heavy chains of the completed intracellular IgG molecule began with pyrrolidonecarboxylic acid after both 1 and 5h of labelling. (4) The IgG, after secretion by plasmacytoma Adj PC5, was found to have the sequence [unk]Glu- Val-Gln-Leu- at the N-termini of the heavy chains.     

Sequence studies of the Fd section of the heavy chain of rabbit immunoglobulin G

A partial amino acid sequence was given by Cebra, Steiner & Porter (1968b) of the N-terminal half of the heavy chain of rabbit immunoglobulin G. This was extended and in part corrected to give a continuous sequence of 136 residues, which together with other work accounts for three-quarters of the total sequence. Evidence is given suggesting that there is a limited region of 10–15 residues that are exceptionally variable in the heavy chains from pooled rabbit immunoglobulin G.     

A blocked N-terminal residue in the light chain of rabbit immunoglobulin G

The light chain of rabbit immunoglobulin G was shown to contain 15-20% blocked N-terminal residue. The blocked residue is pyrrolid-2-one-5-carboxylic acid, and most of the chains that contain this residue have the N-terminal sequence pyrrolid-2-one-5-carbonyl-valine.     

Gene for an immunoglobulin-binding protein from a group G streptococcus.

The gene (spg) for an immunoglobulin G (IgG)-binding protein from a Streptococcus clinical isolate of Lancefield group G was cloned and expressed in Escherichia coli. The complete nucleotide sequence of the gene and 5'-flanking sequences was determined. The DNA sequence includes an open reading frame which encodes a hypothetical protein of 448 amino acid residues (Mr = 47,595). The 5' end of this open reading frame encodes a sequence resembling a typical secretion signal sequence, and the remainder of the encoded protein has features reminiscent of staphylococcal protein A and of streptococcal M6 protein, including repeated sequences and a similar C-terminal structure. Aside from this C-terminal structure, the encoded protein has little direct amino acid sequence homology to either protein A or M6 protein. In E. coli, the cloned gene directs the synthesis of a protein which binds to immunoglobulins, including rabbit immunoglobulin, goat IgG, and human IgG3(lambda). Its binding properties are similar to those of the protein G described by Bj?rck and Kronvall (L. Bj?rck and G. Kronvall, J. Immunol. 133:969-974, 1984), a type III Fc receptor from a group G streptococcus.     

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.     

Molecular stability of chicken and rabbit immunoglobulin G.

Molecular stability of chicken egg yolk immunoglobulin G (IgY) and that of rabbit IgG were compared by measuring antibody activities and conformational changes. Stability of rabbit IgG to acid denaturation was much higher than that of IgY. Conformation of the IgY molecule was readily changed in acidic conditions, resulting in a rapid loss of antibody activity. Much less stable natures of IgY to heat-treatment and guanidine-HCl denaturation than rabbit IgG were also observed. Differences in the structure between the two immunoglobulins that might participate in their different stability were inferred from their amino acid sequence data. Importance of the intramolecular disulfide linkage in the rabbit light chain and some other structural differences were suggested.     

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.     

Identification,characterization, and cloning of an immunoglobulin degrading enzyme in the cat flea,Ctenocephalides felis

The degradation of cat immunoglobulin G (IgG) in blood-fed adult C. felis midguts was examined. SDS-PAGE analysis of dissected midgut extracts obtained from C. felis that had been blood fed for various times between 0 to 44 h revealed that by 24 h most of the high molecular weight proteins, including the heavy chain of IgG, were digested. A 31-kDa serine protease with IgG degrading activity was purified from fed C. felis midguts by benzamidine affinity chromatography, hydrophobic interaction chromatography, and cation exchange chromatography. Three primary cleavage products between 30- and 40-kDa were observed when the purified protease was incubated with protein A purified cat IgG. N-terminal amino acid sequence analysis of the products revealed that the IgG degrading protease cleaves after specific cysteine and lysine residues within the hinge region of IgG. The enzyme is also capable of degrading other immunoglobulins, serum albumin, and hemoglobin, suggesting that it may have roles in both combating the host's immune system and providing nutrients for the flea. A cDNA clone encoding the 265 amino acid IgG degrading protease proenzyme was isolated. When expressed in a baculovirus/insect cell expression system, the recombinant protein had the same N-terminus as the processed 237 amino acid mature native protein and possessed IgG degrading activity indistinguishable from the native protein. Arch. Insect Biochem.     

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.