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. 2008 Oct;76(10):4530-7.
doi: 10.1128/IAI.00186-08. Epub 2008 Aug 4.

Amylase-binding protein B of Streptococcus gordonii is an extracellular dipeptidyl-peptidase

Biswendu Chaudhuri  1 , Susanna Paju, Elaine M Haase, M Margaret Vickerman, Jason M Tanzer, Frank A Scannapieco
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V体育平台登录 - Amylase-binding protein B of Streptococcus gordonii is an extracellular dipeptidyl-peptidase

"VSports手机版" Biswendu Chaudhuri et al. Infect Immun. 2008 Oct.

Abstract

The oral commensal bacterium Streptococcus gordonii interacts with salivary amylase via two amylase-binding proteins, AbpA and AbpB. Based on sequence analysis, the 20-kDa AbpA protein is unique to S. gordonii, whereas the 82-kDa AbpB protein appears to share sequence homology with other bacterial dipeptidases. The aim of this study was to verify the peptidase activity of AbpB and further explore its potential functions. The abpB gene was cloned, and histidine-tagged AbpB (His-AbpB) was expressed in Escherichia coli and purified VSports手机版. Its amylase-binding activity was verified in an amylase ligand binding assay, and its cross-reactivity was verified with an anti-AbpB antibody. Both recombinant His-AbpB and partially purified native AbpB displayed dipeptidase activity and degraded human type VI collagen and fibrinogen, but not salivary amylase. Salivary amylase precipitates not only AbpA and AbpB but also glucosyltransferase G (Gtf-G) from S. gordonii supernatants. Since Streptococcus mutans also releases Gtf enzymes that could also be involved in multispecies plaque interactions, the effect of S. gordonii AbpB on S. mutans Gtf-B activity was also tested. Salivary amylase and/or His-AbpB caused a 1. 4- to 2-fold increase of S. mutans Gtf-B sucrase activity and a 3- to 6-fold increase in transferase activity. An enzyme-linked immunosorbent assay verified the interaction of His-AbpB and amylase with Gtf-B. In summary, AbpB demonstrates proteolytic activity and interacts with and modulates Gtf activity. These activities may help explain the crucial role AbpB appears to play in S. gordonii oral colonization. .

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Figures

FIG. 1.
FIG. 1.
Cloning, purification, and characterization of AbpB. (A) Linear diagram of p218-1, expressing the His-AbpB fusion protein. T7-p, T7 promoter; T7-t, T7 terminator; lacO, lac operator; LIC site, ligation-independent cloning site; rbs, ribosome binding site; ORF, open reading frame. (B) SDS-PAGE of lysate fractions showing the expression of His-AbpB in E. coli harboring p218-1. Lane 1, molecular mass standard in kDa; lanes 2 and 3, Coomassie blue staining of soluble inclusion bodies and supernatant of IPTG-induced E. coli cells, respectively; lanes 4 and 5, Coomassie blue staining of 4 and 2 mg of purified protein, respectively; lanes 6 and 7, amylase ligand binding assay of 400 and 200 ng of His-AbpB, respectively.
FIG. 2.
FIG. 2.
Purification of native AbpB, zymography, enzymatic assay, and cleavage of host proteins by AbpB. (A) Protein elution profile from Bio-Gel P-60 molecular exclusion column. Fractions of 5 ml were collected. Only fractions 3 and 4 displayed peptidase activity. (B) SDS-PAGE analysis of pooled fractions 3 and 4 from Bio-Gel P-60 containing peptidase activity is shown with Coomassie blue staining. Lanes 1 and 2 contained 10 and 5 μg protein, respectively. (C) Amylase ligand binding assay of native, enriched AbpB (5 μg protein). (D) Western blot using anti-AbpB antibody with native, enriched AbpB (5 μg protein). (E) Gelatin zymography of native AbpB and His-AbpB. Lane 1, boiled AbpB (5 μg); lane 2, AbpB (5 μg); lane 3, boiled His-AbpB (4 μg); lane 4, His-AbpB (4 μg); lane 5, protein from E. coli cell extract (control); lane 6, recombinant amylase-binding protein A (rAbpA) as a control; lane 7, molecular mass standard in kDa. (F) Enzymatic assay of AbpB using chromogenic substrates. Release of p-nitroaniline, representing dipeptidase activity, was measured at 405 nm using a microplate reader. Dark bars, results using His-AbpB; white bars, results using native AbpB. (G) Proteolytic cleavage of host proteins by His-AbpB. Substrate proteins (5 μg each) were incubated with 400 ng of His-AbpB at 37°C for 20 h. Cleavage patterns were visualize by Coomassie staining. Lane 1, human collagen VI alone; lane 2, human collagen VI plus His-AbpB; lane 3, salivary amylase alone; lane 4, salivary amylase plus His-AbpB; lane 3, fibrinogen alone; lane 6, fibrinogen with His-AbpB. Arrowheads indicate protein bands in untreated samples degraded following incubation with AbpB.
FIG. 3.
FIG. 3.
Immunoaffinity purification of AbpB. (A) Coomassie blue-stained gel. Lane 1, culture supernatant starting material; lane 2, material eluted from the AbpB affinity column. (B) Amylase-ligand binding assay. Lane 1, culture supernatant starting material; lane 2, material eluted from the AbpB affinity column. (C) Western blot using anti-AbpB antibody. Lane 1, culture supernatant starting material; lane 2, material eluted from the AbpB affinity column.
FIG. 4.
FIG. 4.
Measurement of sucrase and transferase activities of Gtf-B. Purified Gtf-B enzyme was incubated at 37°C overnight with sucrose in the presence or absence of amylase and/or His-AbpB protein. The amounts of glucose and fructose present in the reaction mixtures were measured using the F-Kit. Transferase (A) and sucrase (B) activities of Gtf-B in the presence of amylase and/or His-AbpB protein are shown by the histograms.
FIG. 5.
FIG. 5.
Binding of His-AbpB and amylase with Gtf-B. Four, 2, 1, 0.5, and 0.25 μg of either His-AbpB or amylase were used to coat wells of an ELISA plate. To each well, 0.5 μg of Gtf-B was added. Gray bars indicate binding of Gtf-B protein to immobilized His-AbpB; white bars indicate binding of Gtf-B to immobilized amylase.
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