We have recently shown that the hbp35 gene, which encodes a hemin-binding protein with one thioredoxin motif and a CTD, is transcribed as a monocistronic 1.1-kb mRNA, but it is subsequently translated into three discrete (+)-Butaclamol hydrochloride cytoplasmic proteins with molecular masses of 40, 29 and 27 kDa, and a diffuse cell surface protein with a molecular mass of 50�C 90 kDa. The diffuse HBP35 protein reacts with the monoclonal antibody 1B5, which recognizes a glycan epitope of anionic polysaccharides. These results suggested that the P. gingivalis HBP35 protein, like RgpB, is glycosylated on the cell surface. The antibody mAb 1B5 recognizes a Mana1-2Mana1-phosphate side chain in anionic polysaccharides but not lipopolysaccharides or capsular polysaccharides. Because anionic polysaccharide was found to be linked to a lipid A core, it was recently renamed ALPS. Our previous study showed that the porR gene, encoding a Cinnarizine putative aminotransferase, plays a role in colony pigmentation on blood agar plates and that mAb 1B5 does not recognize any products in the porR mutant, suggesting that porR is involved in the biosynthesis of A-LPS. Thereafter, mutant studies using vimA, vimE, vimF, wbpB, rfa encoding a heptosyl transferase, waaL encoding an O-antigen ligase, wzy encoding an O-antigen polymerase and gtfB have shown that these genes are also involved in A-LPS biosynthesis. However, the mechanisms of A-LPS biosynthesis and of HBP35 protein binding to A-LPS remain to be determined. We found a gene that is responsible for the translocation of gingipains and HagA to the cell surface. Since then, sov and pg27 have been reported to contribute to gingipain secretion. We recently identified 11 genes that are involved in the secretion of gingipains and HagA and are designated the Por secretion system. In this study, we characterized the secretion and glycosylation mechanism of HBP35 in P. gingivlais and found that HBP35 is transported by the PorSS and is glycosylated with A-LPS on the cell surface. We then determined whether HBP35 is present on the surface of PorSSdeficient mutant cells. Dot blot analysis revealed that the intact cells of the 11 PorSS-deficient mutants blotted on a nitrocellulose membrane showed very weak reactivity with anti- HBP35 antibodies compared to PorSS-proficient strains. In contrast, the 11 PorSS-deficient mutants showed the same reactivity with anti-A-LPS and anti-prolyl tripeptidyl peptidase A antibodies as PorSS-proficient strains. PtpA, a cell surface protein, is secreted PorSS-independently.