The GPI anchor signal sequence dictates the folding and functionality of the Als5 adhesin from Candida albicans

Candida albicans Als5 (adhesin, smaller allelic variant from strain CAI4), expressed in E. coli as an N-terminal GST fusion with its 20-amino-acid C-terminal GPI attachment signal sequence left uncleaved (GST-Als5-SS), differs in secondary structure from the same protein lacking that signal (GST-Als5). In circular dichroism of the proteins at 0.09 mg/ml (pH 8.0, 25°C, GST spectrum subtracted), CONTIN fits Als5 as β-strand rich (closest solution 25.4% β strand, 9.2% α-helix, 43.8% turn, 21.6% disordered) and Als5-SS as predominantly α-helical (closest solution 73.6% α-helix, 26.4% disordered).

Candida albicans Als5 without its C-terminal GPI attachment signal sequence (GST-Als5, purified from E. coli) adheres more strongly to immobilized human collagen type IV than the same protein carrying the 20-amino-acid signal sequence (GST-Als5-SS). Both bind in an amount-of-collagen-dependent manner at 37°C (protein at 0.09 mg/ml, 1 h), and GST alone binds very poorly.

Treating immobilized collagen type IV with 0.5 U N-glycosidase F for 2 hours lowers adhesion of Candida albicans GST-Als5 by roughly 20%, and extending the treatment to 3 hours does not lower it further. Carbohydrate chains of collagen type IV therefore contribute to Als5 adhesion but are not its sole determinant, and non-glycosylated Als5 made in E. coli still binds collagen type IV.

Candida albicans GST-Als5 aggregates extensively after incubation at 37°C for 2 weeks (0.4 µM, TEM), whereas GST-Als5-SS, which carries the 20-amino-acid C-terminal GPI attachment signal sequence, shows very poor aggregation under the same conditions, and GST alone does not aggregate significantly. The aggregation of GST-Als5 is typical of β-sheet-rich proteins.

The GPI attachment signal sequence of Candida albicans Als5 is itself aggregation-prone, since TANGO predicts a high β-aggregation propensity in the C-terminal half of residues 1328–1347 and the synthetic SS peptide (40 µM) aggregates alone at 37°C within 2 days by TEM. GST-Als5 (0.4 µM) still aggregates when incubated with this peptide for 2 days, yet GST-Als5-SS does not aggregate, which indicates that the amyloidogenic regions of Als5-SS are not exposed to solvent as they are in Als5.

Candida albicans GST-Als5 and GST-Als5-SS bind the peptides KLRIPSV, AYKSLMT and EHAHTPR in a temperature-dependent manner, as measured by intrinsic tryptophan fluorescence. Neither protein binds any peptide at 4°C. At 15°C only GST-Als5-SS binds, and only EHAHTPR (Kd about 65 nM). At 27°C GST-Als5-SS binds all three peptides with roughly comparable affinities while GST-Als5 binds none. At 37°C, 47°C and 52°C both proteins bind all three. The reversed-sequence peptide VSPIRLK does not bind either protein at 27°C or 47°C.

Peptide binding by Candida albicans GST-Als5 and GST-Als5-SS (KLRIPSV, AYKSLMT, EHAHTPR) is largely endothermic, strengthens with temperature and becomes more favorable in ΔG°, so it is predominantly hydrophobic and entropy driven. For GST-Als5 binding EHAHTPR, Kd falls from 16.0 ± 3.8 × 10^-8 M at 37°C to 3.4 ± 1.1 × 10^-8 M at 47°C while ΔG° moves from -40.46 to -45.83 kJ/mol.

Both GST-Als5 and GST-Als5-SS from Candida albicans bind the peptide EHAHTPR, although a previous study reported that EHAHTPR does not bind Als5 displayed on the surface of S. cerevisiae from a different C. albicans strain (CA1). This suggests that strain-specific or allelic differences can change the peptide specificity of Als5.

In Rosetta homology models of the Als5 N-terminal domain built on the C. albicans Als9-2 apo structure (PDB 2Y7N, 66% identity and 78% similarity), the last 11 residues of the GPI attachment signal sequence (KFISVALFFFL) can take the place of the βG2′ strand that lies over the N1 domain, with or without the peptide ligand EHAHTPR bound. This supports a proposal that, in uncleaved Als5-SS, the signal sequence folds back onto the N-terminal domain, imposes a more structured, α-helical C-terminal domain, and buries the amyloidogenic region (residues 325–329), and that cleavage of the signal releases this constraint.

PONDR-Fit predicts that about 70% of Candida albicans Als5, located in its C-terminal half, is intrinsically disordered. The CD spectrum of Als5 lacking its GPI attachment signal closely resembles that of intrinsic pre-molten globules.

Candida albicans Als5, a Ser/Thr-rich adhesin, migrates by SDS-PAGE at about 270 kDa as a GST fusion in both forms (GST-Als5 and GST-Als5-SS) against expected masses of about 166 and 168 kDa, and intact-mass MALDI-TOF of GST-Als5-SS gives 168.6 kDa. Expressed in E. coli (induction at 16°C for 6 h with 0.1 mM IPTG) the fusions reach only about 0.09 mg/ml, GST-Als5 aggregates and precipitates when concentrated, and the PreScission protease site between GST and Als5 is cleaved poorly.