Figure 1.
C. raphigera β-glucosidase, D2, is encoded by a single gene.
(A) cDNA and amino acid sequences of C. raphigera D2. Upper case DNA sequence indicates the region used as a Southern blotting probe (738–1025bp). Amino acid sequence shown in lower case letters indicates putative signal peptide. Solid box, consensus amino acids for a predicted N-glycosylation site (box with thicker lines indicates the most probable N-glycosylation site). Dotted ovals, common peptides identified by MS in both large and small native D2. (B) Southern blotting of C. raphigera genomic DNA. Left, C. raphigera genomic DNA was cut with indicated restriction enzymes and probed with a PCR-amplified region using genomic DNA as a template. Right, C. raphigera genomic DNA was cut with indicated restriction enzymes and probed with a PCR-amplified region using cDNA as a template.
Figure 2.
C. raphigera produces two variants of β—glucosidase that can be separated by size exclusion chromatography.
The peak of activity recovered from anion exchange chromatography was concentrated and resolved by size exclusion chromatography. BGL activity of the fractions was monitored using pNPG (closed squares) and cellobiose assays (open circles). Two peaks, large (L) and small (S) were identified in varying amounts depending on fungal growth conditions. (A) Sephadex G200 fractions analyzed by pNPG and cellobiose assays from 3-day old YP liquid culture with 14-day old C. raphigera on potato dextrose agar with agar also transferred into the liquid medium. (B) From 3-day old YP liquid medium with 10 mM cellobiose with 14-day old C. raphigera on potato dextrose agar. (C) From YP liquid medium with 10 mM cellobiose inoculated with spores scraped from a 14-day old C. raphigera plate. Results were representative of three independent studies.
Figure 3.
Protein bands corresponding to BGL activities identified as D2 by MS.
Concentrated size exclusion chromatography fraction pools were run on a 7% SDS polyacrylamide gel and stained with SYPRO Ruby protein stain overnight, and photographed using Gel Doc system (Bio-Rad). L: higher molecular weight peak, S: lower molecular weight peak. For the actual sample submission, L and S were loaded 2 lanes apart from each other to avoid cross-contamination before cutting out the bands. None of the bands was identified with >95% probability as a known protein in NCBI database using Mascot sequence query at the time of analysis (December 2010). All 3 bands were identified as the D2 protein with 100% probability.
Figure 4.
rD2 and nD2S are N-glycosylated, but nD2L has more extensive modification besides N-glycans.
(A) D2 variants on a 7% native polyacrylamide gel. Left: SYPRO Ruby protein staining, middle: MUG BGL activity staining, right: merged image of MUG and SYPRO Ruby stainings. Protein ladder did not clearly separate according to a correct size pattern on a native gel and thus omitted. (B) SYPRO Ruby-staining of D2 variants, boiled and run on a 7% SDS gel, with or without EndoH treatment. PageRuler Unstained (Fermentas) was used as a size marker.
Figure 5.
MALDI/TOF-MS spectra shows O-glycosylation of nD2L.
O-linked profiling of equal amounts of nD2L and nD2s protein harvested indicated that nD2L is heavily O-glycosylated (A), while no O-glycosylation was detected on nD2S (B). Signals shown with asterisks (*) are background signals from the matrix. A signal shown with a diamond (⧫) denotes the presence of a small glycan fragment (correspond to Hexose1Deoxyhexose1, non-reduced form).
Figure 6.
Fragmentation of deoxyhexose-containing O-glycans in nD2L.
The main neutral loss observed in MS/MS from deoxyhexose-containing O-glycans was loss of m/z 189, indicating the deoxyhexose located at non-reducing end (A and D). The neutral loss of m/z 189 in MS2, followed by the loss of m/z 174 in MS3 in E, shows the presence of internal deoxyhexyose (m/z 174), indicating sequential terminal deoxyhexose residues. A fragment ion m/z 275 (C) and the neutral loss of m/z 252 (F) demonstrate that there is no branching at the reducing-end hexose. Triangle, deoxyhexose; circle, hexose; gray shapes indicate lost residues.
Figure 7.
nD2L does not exhibit substrate inhibition by pNPG.
pNPG assay was performed as described in Materials and Methods. The Lineweaver-Burk plot shows a substrate inhibition curve (emphasized in the inset) that indicates diminishing activity of nD2L and rD2 as substrate concentration increases.
Figure 8.
nD2L is more active toward cellobiose than small forms of D2.
Maximum activity ratio of D2 variants toward cellobiose and pNPG. Activity data were complied from at least 3 independent experments. The difference between nD2L and nD2S is statistically significant (p <0.0002) by Welch's t-test. Error bars indicate standard error of the mean.
Table 1.
Km and Ki of partially purified BGLs.
Figure 9.
Glucose inhibition of nD2L in pNPG assay is noncompetitive.
Lineweaver-Burk plots of pNPG activity of D2 variants in the presence of glucose. (A) nD2L, (B) nD2S, (C) rD2. Open symbols: 0 mM, light gray: 3 mM, dark gray: 6 mM, black: 9 mM glucose.
Figure 10.
nD2L is more thermostable than smaller forms at pH 5.0, and nD2L purified from spore-inoculated culture (even more extensively glycosylated) is thermostable at pH 3.0.
pNPG activity of D2 variants that were incubated at 60°C at pH 5.0 or pH 3.0 for indicated length of time. pl designates samples purified from plate-inoculated medium; sp designates samples purified from spore-inoculated medium.
Figure 11.
nD2L is resistant to deactivation by SDS at room temperature.
Relative pNPG activity of D2 variants in the presence of SDS. (A) Enzymes assayed in the presence of SDS without the 1.5-h SDS pre-incubation prior to the assay. (B) Enzymes assayed after incubated in 1× Tris-glycine buffer with 0% (dark gray columns), 0.1% (gray columns) or 1% SDS (light gray columns) for 1.5 h at room temperature.
Table 2.
Summary of enzymatic properties of D2 variants.