The native 67-kilodalton minor fimbria of Porphyromonas gingivalis is a novel glycoprotein with DC-SIGN-targeting motifs

Abstract

We recently reported that the oral mucosal pathogen Porphyromonas gingivalis, through its 67-kDa Mfa1 (minor) fimbria, targets the C-type lectin receptor DC-SIGN for invasion and persistence within human monocyte-derived dendritic cells (DCs). The DCs respond by inducing an immunosuppressive and Th2-biased CD4(+) T-cell response. We have now purified the native minor fimbria by ion-exchange chromatography and sequenced the fimbria by tandem mass spectrometry (MS/MS), confirming its identity and revealing two putative N-glycosylation motifs as well as numerous putative O-glycosylation sites. We further show that the minor fimbria is glycosylated by ProQ staining and that glycosylation is partially removed by treatment with beta(1-4)-galactosidase, but not by classic N- and O-linked deglycosidases. Further monosaccharide analysis by gas chromatography-mass spectrometry (GC-MS) confirmed that the minor fimbria contains the DC-SIGN-targeting carbohydrates fucose (1.35 nmol/mg), mannose (2.68 nmol/mg), N-acetylglucosamine (2.27 nmol/mg), and N-acetylgalactosamine (0.652 nmol/mg). Analysis by transmission electron microscopy revealed that the minor fimbria forms fibers approximately 200 nm in length that could be involved in targeting or cross-linking DC-SIGN. These findings shed further light on molecular mechanisms of invasion and immunosuppression by this unique mucosal pathogen.

Figures

FIG. 1.

Purification and characterization of the minor (Mfa1) fimbria. (A) Elution profile of the 67-kDa fimbria on DEAE-Sepharose CL-6B, showing a peak that eluted with 0.3 M NaCl. (B) SDS-PAGE analysis of the minor fimbria. Lane 1, MW standard; lane 2, Coomassie blue stain showing the 67-kDa minor fimbria; lane 3, silver stain showing the single band of the minor fimbria (arrow). (C) Transmission electron micrograph of the purified minor fimbria showing 100- to 200-nm fibers. Bar, 100 nm.

FIG. 2.

Glycosylation of the minor fimbriae. (A) Peptide sequence obtained by MS/MS (shown in bold), which confirmed the identity of the minor fimbria. Boxed are putative N-X-S/T asparagine-linkage motifs. (B) Confirmation of glycosylation on the minor fimbria by ProQ (glycosylation stain). Minor fimbriae samples were run on SDS-PAGE gels and stained with ProQ (lanes 1 to 3), and then the same gel was stained with Coomassie (lanes 4 to 6). Lanes 1 and 4, nonglycosylated MW standard; lanes 2 and 5, CandyCane glycoprotein standard; lanes 3 and 6, the minor fimbria. White arrowheads highlight the CandyCane (Molecular Probes) glycoprotein standard.

FIG. 3.

Enzymatic deglycosylation of the minor fimbria observed in the presence of endoglycosidase F2, endoglycosidase F3, and β(1-4)-galactosidase. Enzymatic deglycosylation treatment on purified minor fimbria (Mfa1), as verified by lack of shift or the loss of ProQ (glycosylation detection) signal, is shown. (A, C, E) ProQ gels; (B, D, and F) the same gel after Coomassie blue staining. (A and B) Nonreduced native fimbria treated with endoglycosidase. All lanes were loaded with 5 μg of Mfa1 and digested with the indicated endoglycosidase(s). (C and D) Fimbriae denatured prior to treatment with endoglycosidase. All lanes were loaded with 7 μg of Mfa1 and digested with the indicated endoglycosidase(s). (E and F) Minor fimbria pretreated with α-l-fucosidase, then denatured and treated with endoglycosidase. All lanes were loaded with 7 μg Mfa1 and digested with the indicated endoglycosidase(s).

FIG. 4.

Representative chromatograph from GC-MS analysis of the purified minor fimbria. The purified minor fimbria was analyzed by GC-MS for monosaccharide content relative to monosaccharide standards for Fuc, Xyl, Man, Gal, Glc, GalNac, and GlcNAc.