Expression of Transglutaminase Substrate Activity on Candida albicans Germ Tubes through a Coiled, Disulfide-bonded N-terminal Domain of Hwp1 Requires C-terminal Glycosylphosphatidylinositol Modification

Janet F Staab,Yong-Sun Bahn,Chia-Hui Tai,Paul F Cook,Paula Sundstrom

doi:10.1074/jbc.m406005200

Abstract

By serving as a microbial substrate for epithelial cell transglutaminase, Hwp1 (Hyphal wall protein 1) of Candida albicans participates in cross-links with proteins on the mammalian mucosa. Biophysical properties of the transglutaminase substrate domain were explored using a recombinant protein representative of the N-terminal domain of Hwp1 and were similar to other transglutaminase substrates, the small proline-rich proteins of cornified envelopes found in stratified squamous epithelia. Recombinant Hwp1 lacks alpha and beta structures by circular dichroism and likely exists as a disulfide-cross-linked coiled-coil. The transglutaminase substrate property prompted a unique approach for investigating the features of surface Hwp1 on germ tubes. A lysine analog, 5-(biotinamido)pentylamine, was cross-linked to germ tubes catalyzed by transglutaminase 2 prior to cell fractionation, immunoprecipitation, and detection with streptavidin conjugates. The majority of the transglutaminase-modifiable Hwp1 was covalently attached to the beta-glucan of hyphae by the C terminus of Hwp1 via a glycosylphosphatidylinositol remnant anchor. A putative precursor of cell wall forms of Hwp1 was identified in the cell extract and in the culture medium. Hwp1 was modified by relatively short N-linked glycans, and the molecular size of the protein was reduced by hypomannosylation when expressed in O-glycosylation mutant strains. Hwp1 combines features of mammalian transglutaminase substrate proteins with characteristics of fungal cell wall proteins to form an unconventional adhesin at the hyphal wall of C. albicans.

Highlights

¶ Present address: Depts. of Molecular Genetics and Microbiology, Duke University Medical Center, 315 CARL Bldg., Research Dr, Durham, NC 27710
Role of the N Terminus of Hwp1 in Adherence of C. albicans to Buccal Epithelial Cells—The N-terminal domain of Hwp1 is composed of an acidic, degenerate amino acid repeat rich in proline and glutamine amino acids and is notably deficient in glycosylation sites [23]
The function of the N terminus of Hwp1 in stabilized adhesion to the surface of buccal epithelial cells (BECs) was investigated by using rHwp1N13, comprising amino acids 1–148 of the mature protein as a competitor in adhesion assays [10]

Summary

EXPERIMENTAL PROCEDURES

C. albicans Strains—C. albicans strains SC5314 (wild type) [14], CAH7–1A (hwp1/hwp null mutant), and CAH7–1A1 (hwp1/hwp null mutant, UraϪ strain) [10] were used in experiments to identify Hwp. The Sepharose beads were washed as before [19] and suspended in 25 ␮l of sample buffer (Invitrogen/Novex Tris-Acetate PAGE system), heated to 70 °C for 10 min, spun, and the supernatants were transferred to new tubes. The HF-released Hwp was examined directly by adding 1ϫ sample buffer to the methanol-washed, dried cell walls, heating to 70 °C for 10 min and analyzing samples of the supernatants by SDSPAGE and Western blotting. The protein pellets were washed with acetone, dried, suspended in 1ϫ sample buffer, heated to 70 °C, and analyzed by SDS-PAGE and Western blotting. The PI-PLC and SDS lysis bufferreleased proteins from the cell walls were diluted 1:3 and 1:4, respectively, in radioimmune precipitation assay buffer [18] before adding 20 ␮l of polyclonal rabbit anti-rHwp1N13 serum.

RESULTS

Subcellular fraction

DISCUSSION