Secretion of cryparin, a fungal hydrophobin

Abstract

Cryparin is a cell-surface-associated hydrophobin of the filamentous ascomycete Cryphonectria parasitica. This protein contains a signal peptide that directs it to the vesicle-mediated secretory pathway. We detected a glycosylated form of cryparin in a secretory vesicle fraction, but secreted forms of this protein are not glycosylated. This glycosylation occurred in the proprotein region, which is cleaved during maturation by a Kex2-like serine protease, leaving a mature form of cryparin that could be isolated from both the cell wall and culture medium. Pulse-chase labeling experiments showed that cryparin was secreted through the cell wall, without being bound, into the culture medium. The secreted protein then binds to the cell walls of C. parasitica, where it remains. Binding of cryparin to the cell wall occurred in submerged culture, presumably because of the lectin-like properties unique to this hydrophobin. Thus, the binding of this hydrophobin to the cell wall is different from that of other hydrophobins which are reported to require a hydrophobic-hydrophilic interface for assembly.

FIG. 1

Pulse-chase analysis of C. parasitica EP155. A 30-min pulse of [³⁵S]cysteine was added to a 3-day old liquid culture of the fungus. Samples were taken at the indicated time points (in minutes) following removal of the radiolabel from the culture medium. Cryparin was extracted from the lyophilized cell walls or culture fluid and electrophoresed on 12% polyacrylamide SDS-PAGE gel. The gel was dried and exposed to a phosphorimager screen for 24 h. (A) Labeled cryparin in cells. (B) Labeled cryparin in the culture medium.

FIG. 2

Time course of relative distribution of pulse-labeled cryparin. Samples from the pulse-chase analysis (Fig. 1) were quantified with a phosphorimager. Units are arbitrary phosphorimager units. ●, ³⁵S-cryparin isolated from cell walls at different times following the chase with unlabeled cysteine; ○, ³⁵S-cryparin isolated from culture medium at these times; ▾, total ³⁵S detected in cryparin in cell walls and culture fluid at each time. The data shown are representative of multiple experiments. These could not all be shown, because the quantitative counts are different for each experiment, but the pattern of labeling is the same in all cases.

FIG. 3

Binding of cryparin to the cell surface of a C. parasitica strain from which cryparin had been genetically deleted. Cryparin was extracted from the cell walls of C. parasitica strains and detected by PAGE. Lanes: M, protein size standards; 1, cryparin extracted from 3-day-old mycelia of EP155; 2, cryparin extracted from 3-day-old mycelia of the cryparin deletion strain Δ119, which had been incubated with the culture fluid of EP155 for 4 h followed by two washes with water.

FIG. 4

Growth rate of C. parasitica EP155 and incremental cryparin accumulation in liquid culture. ●, dry weight of mycelia (grams) in 100 ml of EP complete liquid medium. ■, amount of cryparin produced per gram of mycelia in the first 5 h of a 24-h period. For each day, a pulse of [³⁵S]cysteine was added to a new flask of 100 ml of EP complete liquid culture of the fungus that had been growing for the indicated time. Samples were taken 5 h after addition of the radiolabel. Cryparin was extracted from the lyophilized mycelium and separated by 12% polyacrylamide SDS-PAGE. The gel was dried and exposed to a phosphorimager screen for 24 h, and the total amount of cryparin was measured.

FIG. 5

Presence of cryparin in a vesicle fraction. Vesicles were isolated by polyethylene glycol precipitation and differential centrifugation from strain EP155 and the isogenic strain Δ119 from which the cryparin gene had been deleted (Δcrp). (A) Silver-stained gel of vesicle proteins. (B) Corresponding Western blot with antibody against cryparin.

FIG. 6

Evidence for a high-molecular-mass glycosylated intermediate form of cryparin in cells. (A) Western blot of cryparin purified from culture medium (M) and C. parasitica whole cells (i.e., cell wall and cytoplasm [C]). Cryparin was detected with a polyclonal antibody to cryparin. (B) Detection of glycosylated cryparin by using the Glycotrack carbohydrate detection kit. Only the cryparin extracted from mycelium was shown to possess carbohydrate residues.