Characterization of Wheat Germ Agglutinin Lectin-Reactive Glycosylated OmpA-Like Proteins Derived from Porphyromonas gingivalis

Abstract

ABSTRACTGlycosylation is one of the common posttranslational modifications in eukaryotes. Recently, glycosylated proteins have also been identified in prokaryotes. A few glycosylated proteins, including gingipains, have been identified inPorphyromonas gingivalis, a major pathogen associated with chronic periodontitis. However, no other glycosylated proteins have been found. The present study identified glycoproteins inP. gingivaliscell lysates by lectin blotting. Whole-cell lysates reacted with concanavalin A (ConA),Lens culinarisagglutinin (LCA),Phaseolus vulgariserythroagglutinin (PHA-E4), and wheat germ agglutinin (WGA), suggesting the presence of mannose-,N-acetylgalactosamine-, orN-acetylglucosamine (GlcNAc)-modified proteins. Next, glycoproteins were isolated by ConA-, LCA-, PHA-E4-, or WGA-conjugated lectin affinity chromatography although specific proteins were enriched only by the WGA column. Mass spectrometry analysis showed that an OmpA-like, heterotrimeric complex formed by Pgm6 and Pgm7 (Pgm6/7) was the major glycoprotein isolated fromP. gingivalis. Deglycosylation experiments and Western blotting with a specific antibody indicated that Pgm6/7 was modified withO-GlcNAc. When whole-cell lysates fromP. gingivalismutant strains with deletions of Pgm6 and Pgm7 were applied to a WGA column, homotrimeric Pgm7, but not Pgm6, was isolated. Heterotrimeric Pgm6/7 had the strongest affinity for fibronectin of all the extracellular proteins tested, whereas homotrimeric Pgm7 showed reduced binding activity. These findings suggest that the heterotrimeric structure is important for the biological activity of glycosylated WGA-binding OmpA-like proteins inP. gingivalis.