Interaction between Two Putative Glycosyltransferases Is Required for Glycosylation of a Serine-Rich Streptococcal Adhesin
0301 basic medicine
Glycosylation
Blotting, Western
Genetic Complementation Test
Molecular Sequence Data
Glycosyltransferases
Recombinant Proteins
Acetylglucosamine
3. Good health
Isoenzymes
Mutagenesis, Insertional
03 medical and health sciences
Phenotype
Bacterial Proteins
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Mutation
Escherichia coli
Serine
Amino Acid Sequence
Fimbriae Proteins
Chromatography, Liquid
Plasmids
Protein Binding
DOI:
10.1128/jb.01078-07
Publication Date:
2007-12-15T01:53:31Z
AUTHORS (7)
ABSTRACT
ABSTRACT
Fap1, a serine-rich glycoprotein, is essential for fimbrial biogenesis and biofilm formation of
Streptococcus parasanguinis
(formerly
S. parasanguis
). Fap1-like proteins are conserved in many streptococci and staphylococci and have been implicated in bacterial virulence. Fap1 contains two serine-rich repeat regions that are modified by O-linked glycosylation. A seven-gene cluster has been identified, and this cluster is implicated in Fap1 biogenesis. In this study, we investigated the initial step of Fap1 glycosylation by using a recombinant Fap1 as a model. This recombinant molecule has the same monosaccharide composition profile as the native Fap1 protein. Glycosyl linkage analyses indicated that
N
-acetylglucosamine (GlcNAc) is among the first group of sugar residues transferred to the Fap1 peptide. Two putative glycosyltransferases, Gtf1 and Gtf2, were essential for the glycosylation of Fap1 with GlcNAc-containing oligosaccharide(s) in both
S. parasanguinis
as well as in the Fap1 glycosylation system in
Escherichia coli
. Yeast two-hybrid analysis as well as in vitro and in vivo glutathione
S
-transferase pull-down assays demonstrated the two putative glycosyltransferases interacted with each other. The interaction domain was mapped to an N-terminal region of Gtf1 that was required for the Fap1 glycosylation. The data in this study suggested that the formation of the Gtf1 and Gtf2 complex was required for the initiation of the Fap1 glycosylation and that the N-terminal region of Gtf1 was necessary.
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