Cleavage of bacterial flagellin with cyanogen bromide. Antigenic properties of the protein fragments

Parish, Christopher R.; Wistar, Richard; Ada, G. L.

doi:10.1042/bj1130501

Cited by 43 publications

(22 citation statements)

References 8 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…3, there emerges a strong correlation between the ability of a mAb to bind to a peptide derived from the globular protein antigen, rat cyt c, and the specificity of that mAb for a denatured form of the protein. Antibodies (26,27). In fact, this distinction was the basis for the early classification of epitopes as either sequential or conformational (28).…”

Section: Discussionmentioning

confidence: 99%

Antigenicity and native structure of globular proteins: low frequency of peptide reactive antibodies.

Jemmerson

1987

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Recent reports that peptides can frequently mimic epitopes on globular proteins are inconsistent with early studies demonstrating that antibodies to native globular proteins generally do not bind peptides. This discrepancy could result from current confusion of two different populations of antibodies in antisera: one reacting with peptides and denatured protein and the other reacting only with the native protein. To test this possibility, several hundred monoclonal antibodies to rat cytochrome c were examiped by ELISA for binding the intact protein and cyanogen bromide-cleaved peptides. Inhibition by soluble native cytochrome c identified which antibodies were specific for the native protein. The vast maJQrity of these antibodies did not bind the peptides, whereas most of the antibodies specific for denatured forms did bind them. The results are consistent with the idea that antibodies to denatured antigen are readily detected in solid-phase assays, where some antigen molecules denature as they attach to microtiter plates, and show that these antibodies are generally the ones that react with peptides. Thus, reevaluation of data suggesting that anti-native globular protein antibodies bind peptides is warranted.

show abstract

Section: Discussionmentioning

confidence: 99%

Antigenicity and native structure of globular proteins: low frequency of peptide reactive antibodies.

Jemmerson

1987

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…18,000) (6). Precise antigenic studies in the preceding paper (1) have demonstrated that CNBr-digested flagellin has a slightly reduced affinity for anti-flagellin antibodies (Krel = 5.7 X 10-1).…”

Section: Cyanogen Bromide Digest Of Flagellin: Earlier Work In This Lmentioning

confidence: 99%

Immune Response to Chemically Modified Flagellin

Parish

1971

The Journal of Experimental Medicine

Self Cite

172

View full text Add to dashboard Cite

In the preceding paper (1) some chemical, antigenic, and biological properties of a series of acetoacetylated derivatives of Salmonella adelaide flagellin were described. Several chemical and antigenic tests revealed that as flagellin was acetoacetylated to increasing extents there was a steady decline in the affinity of the molecule for anti-flagellin antibodies. This loss in antigenic activity appeared to be accompanied on the one hand by a reduced capacity to initiate antibody formation but on the other hand b y an enhanced ability to induce antibody tolerance. In this paper the antibody tolerance induced by these acetoacetylated flagellins is studied in more detail. Furthermore, the ability of these acetoacetyl derivatives to induce delayed-type hypersensitivity is reported. F r o m these studies it was found that suppression of antibody formation in adult rats was accompanied by enhanced cell-mediated immunity. In fact, it appears that in adult animals antibody formation and cell-mediated immunity m a y well be opposing immunological processes. By contrast, tolerance induced in neonatal rats by acetoacetyl flagellin existed at the level of both humoral and cell-mediated immunity. Some preliminary aspects of this work have been reported elsewhere. 1 Materials and MethodsAnimals.--Randomly bred Wistar rats of either sex were used. Animals were either obtained directly from the Walter and Eliza Hall Institute of Medical Research, Parkville, Australia, or bred in Canberra from Wistar breeders supplied by the Walter and Eliza Hall Institute. Both adult (6-10 wk of age) and neonatal rats were used.Antigens.--Flagellin and polymerized flagellin from S. adelaide (strain SW 1338; H antigen--fg) and flagellin from S. typhimurium (strain SL 871; H antigen--l, 2) were prepared as described previously (2). A cyanogen bromide (CNBr) digest of S. adelaide flagellin was prepared and fractionated into fragment A and a mixture of fragments B, C, and D as de-1 Parish, C. R. 1971. Suppression of antibody formation and concomitant enhancement of cell-mediated immunity by acetoacetylated derivatives of Salmonella flage]lin.

show abstract

“…The flagellar filament is composed of a single protein, flagellin. The flagellin proteins of E. coli and several other species are conserved in their terminal regions, while the central region is variable and carries H-serotype-specific epitopes (9,17,22,39,40). The structure of the Salmonella enterica LT2 flagellum is known from electron microscopy, X-ray fiber diffraction, and X-ray crystallography.…”

mentioning

confidence: 99%

Species-Wide Variation in the Escherichia coli Flagellin (H-Antigen) Gene

et al. 2003

View full text Add to dashboard Cite

Escherichia coli is a clonal species. The best-understood components of its clonal variation are the flagellar (H) and polysaccharide (O) antigens, both well documented since the mid-1930s because of their use in serotyping. Flagellin is the protein subunit of the flagellum that carries H-antigen specificity. We show that 43 of the 54 H-antigen specificities of E. coli map to the flagellin gene at fliC and sequenced all 43 forms and confirmed specificity of each by cloning and expression. This is, to our knowledge, the first time that all known forms of such a highly polymorphic gene have been fully sequenced and characterized for any species. The established distinction between a highly variable central region and more conserved flanking regions is upheld. The sequences fall into two groups, one of which may be derived from the fliC gene of the E. coli/Salmonella enterica common ancestor, the other perhaps obtained by lateral transfer since species divergence. Comparison of sequences revealed that both horizontal DNA transfer and fixation of mutations under diversifying selection pressure contributed to polymorphism in this locus.The O polysaccharide and flagellin are the two major antigens of gram-negative bacteria, also known respectivly as the O and H antigens. Both are highly polymorphic, and Escherichia coli, if one includes the Shigella strains, has 187 O and 53 H forms defined by serology (4,6,15,21). In this study, we show that 43 of the 53 H forms map to the fliC locus and have sequenced all 43 alleles. In some strains the H-antigen phenotype maps to alternative loci, so we cloned, sequenced, and expressed the fliC gene from type strains to relate definitively H-antigen specificity and sequence. These data supplement the genome sequence data of E. coli K-12 and O157:H7 to give more comprehensive genetic information on the species and, in conjunction with the recently published structure (31) of one flagellin form, will allow analysis of the structural basis of the antigenic variation and development of a molecular typing scheme for the H antigen.The bacterial flagellum projects well beyond the surface of the cell and is rotated to provide motive power. The flagellar filament is composed of a single protein, flagellin. The flagellin proteins of E. coli and several other species are conserved in their terminal regions, while the central region is variable and carries H-serotype-specific epitopes (9,17,22,39,40). The structure of the Salmonella enterica LT2 flagellum is known from electron microscopy, X-ray fiber diffraction, and X-ray crystallography. Three domains are recognized (Fig. 1). The conserved terminal segments form the D1 domain located in the center of the flagellum, while the central region of the protein forms two domains (D2 and D3) exposed on the surface (31). The boundaries between D1 and D2 correspond quite well to the boundaries between the central and terminal regions of the protein as determined by alignment of sequences of different forms. However, because we are dealing mostly with...

show abstract

Cleavage of bacterial flagellin with cyanogen bromide. Antigenic properties of the protein fragments

Cited by 43 publications

References 8 publications

Antigenicity and native structure of globular proteins: low frequency of peptide reactive antibodies.

Antigenicity and native structure of globular proteins: low frequency of peptide reactive antibodies.

Immune Response to Chemically Modified Flagellin

Species-Wide Variation in the Escherichia coli Flagellin (H-Antigen) Gene

Contact Info

Product

Resources

About