Linked Life Data

7995943

Source:http://linkedlifedata.com/resource/pubmed/id/7995943

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1
pubmed:dateCreated
1995-1-19
pubmed:databankReference
pubmed:abstractText
We analyzed the structural correlates underlying the insulin-dependent selection of the specific anti-insulin IgG1 kappa mAb13-producing cell clone, derived from a patient with insulin-dependent diabetes mellitus treated with recombinant human insulin. First, we cloned the germ-line genes that putatively gave rise to the expressed VH and V kappa segments and used them to generate the full (unmutated) "germ-line revertant" of the "wild-type" (somatically mutated) mAb13, using recombinant PCR methods and an in vitro human C gamma 1 and C kappa expression system. The full "germ-line revertant" bound insulin specifically and in a dose-saturable fashion, but with a relative avidity (AVrel) more than three-fold lower than that of its wild-type counterpart (Avrel, 1.69 x 10(-8) vs 4.91 x 10(-9) g/microliters). Second, we established, by reassorting wild-type and germ-line revertant forms of the mAb13 VH and V kappa segments, that the increased Avrel for insulin of mAb13 when compared with its full "germ-line revertant" counterpart was entirely dependent on the mutations in the VH not those in the V kappa chain. Third, we determined, by site-directed mutagenesis experiments, that of the three mutations in the mAb13 VH segment (Ser-->Gly, Ser-->Thr, and Ser-->Arg at positions 31, 56, and 58, respectively), only Arg58 was crucial in increasing the mAb13 Avrel (from 1.44 x 10(-8) to 5.14 x 10(-9) g/microliters) and affinity (Kd, from 189 to 59 nM) for insulin. The affinity enhancement mediated by the VH segment Arg58 residue reflected about a threefold decrease in dissociation rate constant (Koff, from 4.92 x 10(-3) to 1.54 x 10(-3) s-1) but not an increase in association rate constant (Kon, from 2.60 x 10(4) to 2.61 x 10(4) M-1 s-1), and it contrasted with the complete loss of insulin binding resulting from the substitution of the VH segment Asn52 by Lys. The present findings suggest that human insulin, a self Ag, has the potential to recruit a natural autoantibody-producing cell precursor expressing a specific surface receptor for Ag in unmutated configuration, and drive it through affinity maturation. They also show that binding of insulin by such a receptor can be enhanced or completely abrogated by a single amino acid change.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
AIM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jan
pubmed:issn
0022-1767
pubmed:author
pubmed:issnType
Print
pubmed:day
1
pubmed:volume
154
pubmed:geneSymbol
J<down>&kgr;</down>, J<down>H</down>, V<down>&kgr;</down>, V<down>H</down>
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
226-38
pubmed:dateRevised
2011-11-17
pubmed:meshHeading
pubmed-meshheading:7995943-Amino Acid Sequence, pubmed-meshheading:7995943-Animals, pubmed-meshheading:7995943-Antibodies, Monoclonal, pubmed-meshheading:7995943-Antibody Diversity, pubmed-meshheading:7995943-Antigen-Antibody Reactions, pubmed-meshheading:7995943-Autoantibodies, pubmed-meshheading:7995943-Autoimmune Diseases, pubmed-meshheading:7995943-Base Sequence, pubmed-meshheading:7995943-Cloning, Molecular, pubmed-meshheading:7995943-Diabetes Mellitus, Type 1, pubmed-meshheading:7995943-Gene Rearrangement, T-Lymphocyte, pubmed-meshheading:7995943-Humans, pubmed-meshheading:7995943-Hybridomas, pubmed-meshheading:7995943-Immunoglobulin G, pubmed-meshheading:7995943-Immunoglobulin Variable Region, pubmed-meshheading:7995943-Immunoglobulin kappa-Chains, pubmed-meshheading:7995943-Insulin, pubmed-meshheading:7995943-Mice, pubmed-meshheading:7995943-Molecular Sequence Data, pubmed-meshheading:7995943-Mutagenesis, Site-Directed, pubmed-meshheading:7995943-Polymerase Chain Reaction, pubmed-meshheading:7995943-Recombinant Fusion Proteins, pubmed-meshheading:7995943-Recombinant Proteins, pubmed-meshheading:7995943-Sequence Alignment, pubmed-meshheading:7995943-Sequence Homology, pubmed-meshheading:7995943-Templates, Genetic
pubmed:year
1995
pubmed:articleTitle
A human anti-insulin IgG autoantibody apparently arises through clonal selection from an insulin-specific "germ-line" natural antibody template. Analysis by V gene segment reassortment and site-directed mutagenesis.
pubmed:affiliation
Department of Pathology, Cornell University Medical College, New York, NY 10021.
pubmed:publicationType
Journal Article, Comparative Study, Research Support, U.S. Gov't, P.H.S.