Hui Joseph P M, White Theresa C, Thibault Pierre
Institute for Biological Sciences, 100 Sussex Drive, Ottawa, Ontario, Canada, K1A 0R6.
Glycobiology. 2002 Dec;12(12):837-49. doi: 10.1093/glycob/cwf089.
Mass spectrometric techniques combined with enzymatic digestions were applied to determine the glycosylation profiles of cellobiohydrolase (CBH II) and endoglucanases (EG I, II) purified from filamentous fungus Trichoderma reesei. Electrospray mass spectrometry (ESMS) analyses of the intact cellulases revealed the microheterogeneity in glycosylation where glycoforms were spaced by hexose units. These analyses indicated that glycosylation accounted for 12-24% of the molecular mass and that microheterogeneity in both N- and O-linked glycans was observed for each glycoprotein. The identification of N-linked attachment sites was carried out by MALDI-TOF and capillary liquid chromatography-ESMS analyses of tryptic digests from each purified cellulase component with and without PNGase F incubation. Potential tryptic glycopeptide candidates were first detected by stepped orifice-voltage scanning and the glycan structure and attachment site were confirmed by tandem mass spectrometry. For purified CBH II, 74% of glycans found on Asn310 were high mannose, predominantly Hex(7-9)GlcNAc(2), whereas the remaining amount was single GlcNAc; Asn289 had 18% single GlcNAc occupancy, and Asn14 remained unoccupied. EG I presented N-linked glycans at two out of the six potential sites. The Asn56 contained a single GlcNAc residue, and Asn182 showed primarily a high-mannose glycan Hex(8)GlcNAc(2) with only 8% being occupied with a single GlcNAc. Finally, EG II presented a single GlcNAc residue at Asn103. It is noteworthy that the presence of a single GlcNAc in all cellulase enzymes investigated and the variability in site occupancy suggest the secretion of an endogenous endo H enzyme in cultures of T. reesei.
将质谱技术与酶消化相结合，用于确定从丝状真菌里氏木霉中纯化得到的纤维二糖水解酶（CBH II）和内切葡聚糖酶（EG I、II）的糖基化谱。对完整纤维素酶进行电喷雾质谱（ESMS）分析，揭示了糖基化的微观异质性，其中糖型以己糖单位间隔排列。这些分析表明，糖基化占分子量的12 - 24%，并且在每种糖蛋白中均观察到N - 连接和O - 连接聚糖的微观异质性。通过对每种纯化的纤维素酶组分经胰蛋白酶消化且分别有无PNGase F孵育后的产物进行基质辅助激光解吸电离飞行时间质谱（MALDI - TOF）和毛细管液相色谱 - ESMS分析，来鉴定N - 连接的附着位点。首先通过阶梯孔电压扫描检测潜在的胰蛋白酶糖肽候选物，然后通过串联质谱确认聚糖结构和附着位点。对于纯化的CBH II，在Asn310上发现的74%的聚糖为高甘露糖型，主要是Hex(7 - 9)GlcNAc(2)，而其余部分为单个GlcNAc；Asn289有18%被单个GlcNAc占据，Asn14未被占据。EG I在六个潜在位点中的两个位点呈现N - 连接聚糖。Asn56含有单个GlcNAc残基，Asn182主要呈现高甘露糖聚糖Hex(8)GlcNAc(2)，仅有8%被单个GlcNAc占据。最后，EG II在Asn103处呈现单个GlcNAc残基。值得注意的是，在所研究的所有纤维素酶中均存在单个GlcNAc以及位点占据情况的变异性，这表明在里氏木霉培养物中分泌了一种内源性内切糖苷酶H。
