Identification and characterization of a tri-partite hydrophobin from Claviceps fusiformis. A novel type of class II hydrophobin.
A new type of hydrophobin is encoded by an abundant mRNA of Claviceps fusiformis. The predicted amino-acid sequence of the protein, dubbed CFTH1, shows a putative signal sequence for secretion, followed by three class II hydrophobin domains each preceded by glycine/asparagine rich regions. SDS/PAGE analysis of 60% ethanol extractions of C. fusiformis mycelia from shaken cultures showed CFTH1 at the 50-55-kDa position. N-terminal sequencing of both untreated mature CFTH1 and of a fragment obtained by trypsin digestion revealed that CFTH1 is not processed between the hydrophobin domains. Mass spectroscopy showed a mass of about 36 500 Da, which is about 1500 Da higher than the mass predicted from the constituent amino acids, indicating post-translational modification but not glycosylation. Purified CFTH1 self-assembled at hydrophilic/hydrophobic interfaces and, after assembly at a water/air interface, it was found to be highly surface active. Antibodies raised against CFTH1 localized the protein in a mucilageous coat surrounding submerged vegetative hyphae in liquid shaken culture and, as a discrete layer of about 10 nm thickness at the surface of aerial hyphae of standing cultures, suggesting a role in the formation of aerial hyphae.
De Vries
O M
OM
Department of Molecular Plant Biology, Groningen Biomolecular Sciences, University of Groningen, Haren, The Netherlands. O.M.H.de.Vries @biol.RUG.NL
Moore
S
S
Arntz
C
C
Wessels
J G
JG
Tudzynski
P
P
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002967:Claviceps / Q000737:chemistry*; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D007700:Kinetics; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D018076:DNA, Complementary; D005656:Fungal Proteins; C119534:TH1 protein, Claviceps fusiformis