The Streptococcus sanguis platelet aggregation-associated protein. Identification and characterization of the minimal platelet-interactive domain.
Streptococcus sanguis expresses a cell wall-bound protein that induces the activation and aggregation of platelets. This platelet aggregation-associated protein (PAAP) contains a collagen-like, platelet-interactive domain within a 23-kDa protein fragment. To isolate the minimal platelet-interactive domain, p23 PAAP was digested with collagenase, and the digest chromatographed to isolate fractions with activity inhibitory to S. sanguis-induced platelet aggregation. The active fraction was then digested with cyanogen bromide, the product chromatographed, and a smaller inhibitory peptide isolated. Finally, this fraction was digested with endoproteinase Lys-C, and the digest fractionated. After each step, inhibitory activity resolved into single chromatographic peaks of 13 kDa (p13 PAAP), 2.7 kDa (p2.7 PAAP), and a minimal 7-mer peptide, respectively. These PAAP fragments showed similar ID50 (19-28 nM), suggesting that each contained a single copy of the platelet-interactive domain. The minimal 7-mer peptide was purified by immunoaffinity chromatography and reverse phase high pressure liquid chromatography. The primary structure was determined to be Pro-Gly-Glu-Gln-Gly-Pro-Lys. This sequence conforms to the predicted structural motif of the platelet-interactive domains of types I and III collagen. This 7-mer peptide is therefore the platelet-interactive domain of the PAAP from S. sanguis. Its structure explains the molecular basis for immunological cross-reactivity and functional similarity to the platelet-interactive domains of collagens.
Erickson
P R
PR
Department of Preventive Sciences, School of Dentistry, University of Minnesota, Minneapolis 55455.
Herzberg
M C
MC
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D017364:Collagenases / Q000378:metabolism; D016384:Consensus Sequence; D003488:Cyanogen Bromide; D006801:Humans; D008666:Metalloendopeptidases / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D010974:Platelet Aggregation / Q000187:drug effects*; D013298:Streptococcus sanguis / Q000737:chemistry*
D000596:Amino Acids; D001426:Bacterial Proteins; D010446:Peptide Fragments; D017364:Collagenases; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase; D003488:Cyanogen Bromide