Porphyromonas gingivalis HagB binds to dendritic cell membrane proteins

Porphyromonas gingivalis hemagglutinin B (HagB) attaches to the surface of human myeloid dendritic cells.  However, the identity of HagB binding proteins on dendritic cell membranes is not known.  Objectives: The objective of this study was to isolate and identify HagB-binding proteins from human myeloid dendritic cell membrane extracts.  Methods: 3.0 X 10⁶ human myeloid dendritic cells were washed and permeabilized to remove cytoplasmic proteins (DUALXtract, DualSystems Biotech. Inc., San Pedro, CA).  The remaining membrane fragments were solubilized with cell lysis buffer (Cell Signaling, Danvers, MA).  Solubilized membrane proteins were mixed with magnetic beads containing immobilized HagB (Dynabeads, Life Technologies, Corp., Carlsbad, CA).  After the beads were washed, affinity purified HagB binding proteins were removed, separated by Two-Dimensional Fluorescence Difference Gel Electrophoresis (2-D DIGE), picked, and sequenced.  Results: Multiple proteins were isolated and identified from magnetic beads containing immobilized HagB.  Many of these proteins are involved in membrane structure and function.  Among these was a receptor that was previously isolated using a HagB affinity column.  This protein, identified as ACVR2 (activin A receptor type II), may be a potential HagB receptor on the dendritic cell surface.  Conclusions: ACVR2 is a cell surface protein spanning the cytoplasmic membrane that appears to bind HagB.  As a ligand, HagB binding to ACVR2 may affect signal transduction contributing to robust chemokine and cytokine responses seen in HagB-treated dendritic cells.  Supported by funds from NIH NIDCR R01 DE014390.