Localization of the binding site for streptococcal protein G on human serum albumin. Identification of a 5.5-kilodalton protein G binding albumin fragment
(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.
(Less)
- author
- Falkenberg, C LU ; Björck, L LU and Akerström, B LU
- organization
- publishing date
- 1992-02-11
- type
- Contribution to journal
- publication status
- published
- subject
- 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
- LU publication?
- yes
- id
- bda2e4d3-768a-4ee4-a38f-adef2786313c
- date added to LUP
- 2019-05-22 10:25:44
- date last changed
- 2024-04-02 04:34:59
@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}},
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}},
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}},
}