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Localization of the binding site for streptococcal protein G on human serum albumin. Identification of a 5.5-kilodalton protein G binding albumin fragment

Falkenberg, C LU ; Björck, L LU and Akerström, B LU (1992) In Biochemistry 31(5). p.7-1451
Abstract

Protein G is a streptococcal cell wall protein with separate and repetitively arranged binding domains for immunoglobulin G (IgG) and human serum albumin (HSA). In this work, the binding of protein G to HSA was studied. The results suggest that a single binding site is present on HSA: the apparent size of the HSA-protein G complex (230 kDa) corresponded to two or three HSA molecules bound to one protein G molecule, and Ouchterlony immunodiffusion did not yield any precipitate between protein G and HSA. HSA was cleaved by pepsin and CNBr into several fragments which were identified by SDS-PAGE and N-terminal amino acid sequencing, and the binding of protein G to the fragments was studied in Western blot experiments. The results indicated... (More)

Protein G is a streptococcal cell wall protein with separate and repetitively arranged binding domains for immunoglobulin G (IgG) and human serum albumin (HSA). In this work, the binding of protein G to HSA was studied. The results suggest that a single binding site is present on HSA: the apparent size of the HSA-protein G complex (230 kDa) corresponded to two or three HSA molecules bound to one protein G molecule, and Ouchterlony immunodiffusion did not yield any precipitate between protein G and HSA. HSA was cleaved by pepsin and CNBr into several fragments which were identified by SDS-PAGE and N-terminal amino acid sequencing, and the binding of protein G to the fragments was studied in Western blot experiments. The results indicated that the binding area was located in disulfide loops 6-8, involving both the second (loop 6) and the third (loops 7 and 8) domain of HSA. One of the protein G binding pepsin fragments, with an apparent molecular mass of 5.5 kDa, located in loops 7 and 8, was isolated and found to completely inhibit the binding between protein G and the intact HSA, again suggesting a single protein G binding site on serum albumin. Reducing the disulfide bonds of HSA, and subsequent alkylation of the half-cystine residues, significantly decreased the affinity for protein G. Protein G bound to albumin from baboon, cat, guinea pig, hamster, hen, horse, man, mouse, and rat, but not to albumin from cow, dog, goat, pig, rabbit, sheep, snake, or turkey.

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keywords
Amino Acid Sequence, Animals, Binding Sites, Cats, Cattle, Cricetinae, Cyanogen Bromide, Dogs, Guinea Pigs, Horses, Humans, Male, Mice, Molecular Sequence Data, Molecular Weight, Nerve Tissue Proteins/chemistry, Papio, Pepsin A/metabolism, Peptide Fragments/chemistry, Peptide Mapping, Protein Binding, Protein Conformation, Rabbits, Rats, Serum Albumin/chemistry, Sheep, Species Specificity, Streptococcus/chemistry, Structure-Activity Relationship, Swine
in
Biochemistry
volume
31
issue
5
pages
7 - 1451
publisher
The American Chemical Society (ACS)
external identifiers
  • scopus:0026509529
  • pmid:1737003
ISSN
0006-2960
DOI
10.1021/bi00120a023
language
English
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yes
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bda2e4d3-768a-4ee4-a38f-adef2786313c
date added to LUP
2019-05-22 10:25:44
date last changed
2021-01-03 09:39:41
@article{bda2e4d3-768a-4ee4-a38f-adef2786313c,
  abstract     = {<p>Protein G is a streptococcal cell wall protein with separate and repetitively arranged binding domains for immunoglobulin G (IgG) and human serum albumin (HSA). In this work, the binding of protein G to HSA was studied. The results suggest that a single binding site is present on HSA: the apparent size of the HSA-protein G complex (230 kDa) corresponded to two or three HSA molecules bound to one protein G molecule, and Ouchterlony immunodiffusion did not yield any precipitate between protein G and HSA. HSA was cleaved by pepsin and CNBr into several fragments which were identified by SDS-PAGE and N-terminal amino acid sequencing, and the binding of protein G to the fragments was studied in Western blot experiments. The results indicated that the binding area was located in disulfide loops 6-8, involving both the second (loop 6) and the third (loops 7 and 8) domain of HSA. One of the protein G binding pepsin fragments, with an apparent molecular mass of 5.5 kDa, located in loops 7 and 8, was isolated and found to completely inhibit the binding between protein G and the intact HSA, again suggesting a single protein G binding site on serum albumin. Reducing the disulfide bonds of HSA, and subsequent alkylation of the half-cystine residues, significantly decreased the affinity for protein G. Protein G bound to albumin from baboon, cat, guinea pig, hamster, hen, horse, man, mouse, and rat, but not to albumin from cow, dog, goat, pig, rabbit, sheep, snake, or turkey.</p>},
  author       = {Falkenberg, C and Björck, L and Akerström, B},
  issn         = {0006-2960},
  language     = {eng},
  month        = {02},
  number       = {5},
  pages        = {7--1451},
  publisher    = {The American Chemical Society (ACS)},
  series       = {Biochemistry},
  title        = {Localization of the binding site for streptococcal protein G on human serum albumin. Identification of a 5.5-kilodalton protein G binding albumin fragment},
  url          = {http://dx.doi.org/10.1021/bi00120a023},
  doi          = {10.1021/bi00120a023},
  volume       = {31},
  year         = {1992},
}