Production of an Endo-β-N-Acetylglucosaminidase Activity Mediates Growth ofEnterococcus faecalis on a High-Mannose-Type Glycoprotein
Production of an Endo-β-N-Acetylglucosaminidase Activity Mediates Growth ofEnterococcus faecalis on a High-Mannose-Type Glycoprotein
复制标题
内切-β-N-乙酰氨基葡萄糖苷酶活性的产生介导粪肠球菌在高甘露糖型糖蛋白上的生长
作者：
Gretta Roberts;E. Tarelli;K. Homer;J. Philpott‐Howard;D. Beighton
摘要粪肠球菌与很高比例的医院感染有关，然而，对这种细菌在体内增殖的能力知之甚少。研究了一类高甘露糖型糖链(MAN5-到MAN9-GlcNAc2)所占据的模式糖蛋白RNase B对粪肠球菌生长的支持作用。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法测定核糖核酸酶B表明，在细菌生长过程中这种糖蛋白的相对分子质量降低。基质辅助激光解吸电离飞行时间(MALDI-TOF)质谱仪的进一步分析表明，这种质量转移是由于所有高甘露糖型糖体被降解为单一的N-乙酰氨基葡萄糖残基。指数生长过程中的高pH阴离子交换层析分析表明，在培养上清液中存在核糖核酸酶B衍生的多糖，这表明存在内切糖苷酶活性。游离多糖的洗脱保留时间与折叠链霉菌β-N-乙酰氨基葡萄糖苷酶H对核糖核酸酶B的作用所产生的保留时间相同。在细菌生长5h后，没有检测到游离多糖，随后我们证明粪肠球菌中存在甘露糖苷酶活性，它从核糖核酸酶B衍生的多糖中释放游离甘露糖。我们认为粪肠球菌对含有高甘露糖型多糖的糖蛋白的这种脱糖作用以及随后对释放的多糖的降解可能对这种医院内病原体在体内的存活和持续起作用。
ABSTRACT Enterococcus faecalis is associated with a high proportion of nosocomial infections; however, little is known of the ability of this organism to proliferate in vivo. The ability of RNase B, a model glycoprotein with a single N-glycosylation site occupied by a family of high-mannose-type glycans (Man5- to Man9-GlcNAc2), to support growth of E. faecalis was investigated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of RNase B demonstrated a reduction in the molecular mass of this glycoprotein during bacterial growth. Further analysis by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry revealed that this mass shift was due to the degradation of all high-mannose-type glycoforms to a single N-linked N-acetylglucosamine residue. High-pH anion-exchange chromatography analysis during exponential growth demonstrated the presence of RNase B-derived glycans in the culture supernatant, indicating the presence of an endoglycosidase activity. The free glycans were eluted with the same retention times as those generated by the action of Streptomyces plicatusendo-β-N-acetylglucosaminidase H on RNase B. The cleavage specificity was confirmed by MALDI-TOF analysis of the free glycans, which showed glycan species containing only oneN-acetylglucosamine residue. No free glycans were detectable after 5 h of bacterial growth, and we have subsequently demonstrated the presence of mannosidase activity in E. faecalis, which releases free mannose from RNase B-derived glycans. We propose that this deglycosylation of glycoproteins containing high-mannose-type glycans and the subsequent degradation of the released glycans by E. faecalis may play a role in the survival and persistence of this nosocomial pathogen in vivo.
DOI：
10.1016/s0021-9258(18)52343-7
发表时间：
1991-01
期刊：
The Journal of biological chemistry
影响因子：
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作者：
Robert B. TrimbleS;A. Tarentino
通讯作者：
Robert B. TrimbleS;A. Tarentino