Amino acid sequence of the variable regions of heavy chains from two idiotypically cross-reactive human IgM anti-.gamma.-globulins of the Wa group

Biochemistry ◽  
1981 ◽  
Vol 20 (20) ◽  
pp. 5822-5830 ◽  
Author(s):  
David W. Andrews ◽  
J. Donald Capra
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Variable Regions ◽  
Heavy Chains

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

1975 ◽  
Vol 151 (2) ◽  
pp. 337-349 ◽  
Author(s):  
D M Pratt ◽  
L E Mole
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Immunoglobulin G ◽  
Constant Region ◽  
Related Sequence ◽  
Variable Regions ◽  
Heavy Chains ◽  
Continuous Sequence ◽  
Rabbit Igg ◽  
Constant Section

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.

scholarly journals Disulphide bridges of the heavy chain of human immunoglobulin G2

1971 ◽  
Vol 121 (2) ◽  
pp. 217-225 ◽  
Author(s):  
C. Milstein ◽  
B. Frangione
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Heavy Chain ◽  
Amino Acid Sequences ◽  
Cysteic Acid ◽  
Partial Reduction ◽  
Variable Part ◽  
One Dimensional ◽  
Heavy Chains ◽  
Dimensional Electrophoresis

Amino acid sequences around the disulphide bridges of the heavy chain of an immunoglobulin of the γ2 subclass have been studied. The protein was digested with pepsin and the digest fractionated by Sephadex. Screening of the eluate by one-dimensional electrophoresis of oxidized and unoxidized samples was used as an assay and pools of fractions were prepared. Identification by diagonal electrophoresis of several inter- and intra-chain disulphide bridges was done on the pooled fractions. The inter-heavy-chain bridged peptide included four cystine residues. Comparison with proteins of other human subclasses indicated that the intrachain bridges identified are the bridges of the invariable section of γ2 heavy chains. The amino acid sequence of one cysteic acid peptide that may have been derived from the variable part of the molecule was determined. Partial reduction followed by carboxymethylation with radioactive iodoacetate of two proteins of the γ2 class showed a number of labelled peptides that could be identified as being related to the inter-chain bonded cystine residues.

scholarly journals Loss of an individual idiotype on chemical modification. A strategy for assigning idiotypic determinants.

1981 ◽  
Vol 153 (5) ◽  
pp. 1275-1285 ◽  
Author(s):  
J Dickerman ◽  
B Clevinger ◽  
B Friedenson
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Chemical Modification ◽  
Heavy Chain ◽  
Light Chains ◽  
Heavy Chains ◽  
Myeloma Proteins ◽  
Low Levels ◽  
The Individual ◽  
Complementary Strategy

Two dextran-binding myeloma proteins, J558 and Hdex 24, which possess the same individual idiotype (IdI) were diazotized to low levels (1-3.3 groups per subunit) with 1-[14C]-p-aminobenzoate. Both proteins lost the IdI idiotype under these conditions with most of the label incorporated on the heavy chains of each protein. When the diazotization ws carried out in the presence of the hapten 1-O-methyl-alpha-D-glucopyranoside the loss of idiotypic reactivity could be prevented for J558 but not for Hdex 24. Under these conditions most of the label was incorporated on the light chains of J558, but on the heavy chains of Hdex 24. For J558, these results show that a major determinant of the individual idiotype is within the hypervariable positions of the heavy chain. For Hdex 24 the determinant being modified is on the heavy chain but not involved in hapten binding. These results are consistent with previous work showing that J558 and Hdex 24 differ in amino acid sequence in the D and the J segments of the heavy chain and offer an alternative and complementary strategy for assigning idiotypic determinants.

The chemical structure of the heavy chains of rabbit and human immunoglobulin G (IgG)

1966 ◽  
Vol 166 (1003) ◽  
pp. 150-158 ◽  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Heavy Chain ◽  
Chemical Structure ◽  
Antibody Specificity ◽  
Heavy Chains ◽  
Amino Terminal ◽  
Disulphide Bonds ◽  
Rabbit Igg ◽  
Terminal Amino

The combining site of an antibody is entirely contained in the papain digest fragments known as Fab (see Cohen, figure 1, this discussion). These are formed from the light chains and the amino terminal half of the heavy chains known as the Fd fragments. Evidence from many laboratories suggests that the Fd fragment contains the combining site, but it is still uncertain whether the light chain is directly involved in the site, or whether it plays a secondary role in stabilizing the structure of the Fd fragment (see Fleischman 1966). We are attempting, therefore, to determine the chemical structure of the Fd fragment. The evidence of Haber (1964) and Whitney & Tanford (1965) showed that the secondary and tertiary structure of the Fab fragment of several rabbit antibodies could be completely disrupted by reduction of all the disulphide bonds in 6 M guanidine, and that, if such fully denatured molecules were allowed to refold under appropriate conditions, there was a small but significant recovery of affinity for the antigen. These experiments suggest that the specificity of the antibody-combining site is determined by the amino acid sequence of the peptide chain and hence that each antibody specificity would be expected to be reflected in a unique sequence in certain sections of the Fd fragment. It was expected that elucidation of the sequence of this part of the heavy chain would be difficult, as it was evident from earlier work on the amino terminal amino acids of immunoglobulin G (IgG) from several species that mixed sequences were to be expected, and that these may be unrelated to antibody specificity. Hence, considerable variability was probable, only part of which was likely to be related to antibody specificity. On the other hand, some sections of constant sequence should be present, as the Fd fragment contains the interchain disulphide bond between the heavy and light chains and possibly a heavy-heavy interchain bond, as well as the allotypic antigenic sites of the rabbit IgG heavy chain (see Oudin, this discussion). All three features are common to all molecules in a pre­paration of IgG from molecules homozygous at the allotypic locus, and hence it would be expected that they would be located in stable sections of amino acid sequence in Fd fragment. The problem has been tackled in two ways. (1) Work has been commenced with a pathological human IgG which is believed to have a single amino acid sequence and hence can be studied by conventional techniques. Homology between the sequences of several proteins from different mammalian species, suggests that the results will be a valuable guide to what may be expected in IgG from other species such as rabbit, from which purified antibodies may be much more easily prepared. In particular we were fortunate in obtaining a patho­logical human protein the heavy chain of which had a blocked amino terminal amino group, as is found in the rabbit IgG heavy chain and hence the sequence of this protein is being studied. (2) There has never been any convincing demonstra­tion, however, that pathological immunoglobulins have antibody activity, and it is possible that this property may be dependent on some special feature not present, or difficult to recognize, in the pathological protein. Work is also being carried out therefore on the sequence of the Fd fragment of rabbit IgG which has been pre­pared from non-immunized rabbits, from rabbits homozygous at the allotypic locus and from purified rabbit antibodies. Mixed amino acid sequences are found in all these preparations. This precludes a straightforward solution of the sequence and hence attention has been directed to the sequences adjacent to known points such as the amino and carboxyl terminals and the interchain disulphide bonds where it is possible to estimate the relative content of different sequences at these fixed points, and to determine if there is any relation to the known variables of antibody specifi­city and allotypy.

scholarly journals Molecular Cloning of the Gene for a Conserved Major Immunoreactive 28-Kilodalton Protein of Ehrlichia canis: a Potential Serodiagnostic Antigen

1999 ◽  
Vol 6 (3) ◽  
pp. 392-399 ◽  
Author(s):  
Jere W. McBride ◽  
Xue-jie Yu ◽  
David H. Walker
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Gene Family ◽  
Signal Sequence ◽  
Ehrlichia Canis ◽  
Variable Regions ◽  
Reading Frame ◽  
Cowdria Ruminantium ◽  
Canine Ehrlichiosis ◽  
Gene Copies

ABSTRACT A gene encoding a 28-kDa protein of Ehrlichia canis was cloned, sequenced, and expressed, and a comparative molecular analysis with homologous genes of E. canis, Cowdria ruminantium, and Ehrlichia chaffeensis was performed. The complete gene has an 834-bp open reading frame encoding a protein of 278 amino acids with a predicted molecular mass of 30.5 kDa. An N-terminal signal sequence was identified, suggesting that the protein undergoes posttranslational modification to a mature 27.7-kDa protein (P28). The E. canis p28 gene has significant nucleic acid and amino acid sequence homologies with the E. chaffeensisouter membrane protein-1 (omp-1) gene family, with theCowdria ruminantium map-1 gene, and with other E. canis 28-kDa-protein genes. Southern blotting revealed the presence of at least two additional homologous p28 gene copies in the E. canis genome, confirming thatp28 is a member of a polymorphic multiple-gene family. Amino acid sequence analysis revealed that E. canis P28 has four variable regions, and it shares similar surface-exposed regions, antigenicity, and T-cell motifs with E. chaffeensis P28. The p28 genes from seven different E. canisisolates were identical, indicating that the gene for this major immunoreactive protein is highly conserved. In addition, reactivity of sera from clinical cases of canine ehrlichiosis with the recombinant P28 demonstrated that the recombinant protein may be a reliable serodiagnostic antigen.

scholarly journals The complete amino acid sequence of a prototype immunoglobulin-λ light-chain-type amyloid-fibril protein AR

1981 ◽  
Vol 195 (3) ◽  
pp. 561-572 ◽  
Author(s):  
K Sletten ◽  
J B Natvig ◽  
G Husby ◽  
J Juul
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Light Chain ◽  
Amyloid Fibril ◽  
Variable Region ◽  
Amino Acid Residues ◽  
Variable Regions ◽  
Light Chain Type ◽  
Amyloid Fibril Protein ◽  
Chain Type

The amino acid sequence of an amyloid-fibril protein of immunoglobulin light-chain type (AL) was elucidated. The sequence determination involved digesting the protein with trypsin, thermolysin and pepsin. The protein was found to consist of 154 amino acid residues and is thus missing about half of the constant region of a light chain. A certain heterogeneity in the length of the polypeptide was observed in the C-terminal region. The amino acid sequence from CDR (complementary-determining region) 1 and FR (framework region) 3 indicated an oligoclonal origin of the protein. By comparing the primary structure of protein AR with other lambda- and even kappa-chains, it was revealed that protein AR had an insertion of two residues of aspartic acid, namely residues 68 and 69, which has not been reported previously in light chains. The overall sequence homology in the variable region showed that protein AR is more similar to V lambda V than to the other subgroups [Kabat, Wu & Bilofsky (1979) Variable regions of Immunoglobulin Chains, Medical Computer Systems, Bolt, Beranek and Newman, Cambridge, MA].

scholarly journals PHYLOGENETICALLY ASSOCIATED RESIDUES WITHIN THE VHIII SUBGROUP OF SEVERAL MAMMALIAN SPECIES

1973 ◽  
Vol 138 (2) ◽  
pp. 410-427 ◽  
Author(s):  
J. Donald Capra ◽  
Richard L. Wasserman ◽  
J. Michael Kehoe
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Mammalian Species ◽  
Antibody Diversity ◽  
Heavy Chains ◽  
Amino Terminal ◽  
Myeloma Proteins ◽  
Sequence Homologies ◽  
Protein Sequencer ◽  
High Degree

Immunoglobulin heavy chains from IgG pools of several mammalian species have been subjected to Edman degradation on an automated protein sequencer. The percentage of unblocked vs. blocked heavy chains was estimated from the yield of the invariant valine in the second position. Further analysis of these unblocked polypeptides unequivocally placed them in the VHIII subgroup on the basis of homology with known human heavy chain sequences. The mammals studied could be divided into three distinct categories on the basis of the distribution of the VHIII subgroup. In several species the VHIII subgroup could not be detected while, in others, virtually all of the heavy chains belonged to this subgroup. Several species had intermediate amounts with the level of the VHIII subgroup restricted to between 19 and 29% of the total pool. Within experimental error, all members of a given order had a similar VHIII subgroup distribution. Further amino acid sequence studies illustrated a high degree of structural homogeneity in the heavy chains of IgG isolated from pooled sera of a number of mammalian species. The very close amino acid sequence homologies of the amino terminal 24 residues of the various pools corroborated conclusions previously obtained using several myeloma proteins from some of these same species. In particular, certain phylogenetically associated residues were identifiable at characteristic positions in the pools in confirmation of their identification in the myeloma proteins. The simplest assumptions would suggest that these findings are more compatible with a pauci-gene than a multi-gene basis for the generation of antibody diversity.

scholarly journals The amino acid sequences of the Fd fragments of two human γ 1 heavy chains

1970 ◽  
Vol 117 (4) ◽  
pp. 641-660 ◽  
Author(s):  
E. M. Press ◽  
N. M. Hogg
Keyword(s):  
Amino Acid ◽  
Aspartic Acid ◽  
Heavy Chain ◽  
Variable Region ◽  
Amino Acid Sequences ◽  
Light Chains ◽  
Aspartic Acid Residue ◽  
Variable Regions ◽  
Heavy Chain Variable Region ◽  
Heavy Chains

The amino acid sequences of the Fd fragments of two human pathological immunoglobulins of the immunoglobulin G1 class are reported. Comparison of the two sequences shows that the heavy-chain variable regions are similar in length to those of the light chains. The existence of heavy chain variable region subgroups is also deduced, from a comparison of these two sequences with those of another γ 1 chain, Eu, a μ chain, Ou, and the partial sequence of a fourth γ 1 chain, Ste. Carbohydrate has been found to be linked to an aspartic acid residue in the variable region of one of the γ 1 chains, Cor.

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

1970 ◽  
Vol 116 (2) ◽  
pp. 249-259 ◽  
Author(s):  
R. G. Fruchter ◽  
S. A. Jackson ◽  
L. E. Mole ◽  
R. R. Porter
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Heavy Chain ◽  
Immunoglobulin G ◽  
Partial Amino Acid Sequence ◽  
Limited Region ◽  
Heavy Chains ◽  
Total Sequence ◽  
Continuous Sequence ◽  
Rabbit Immunoglobulin

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.

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