Kozloff L M, Turner M A, Arellano F
Department of Microbiology, University of California, San Francisco 94143-0404.
J Bacteriol. 1991 Oct;173(20):6528-36. doi: 10.1128/jb.173.20.6528-6536.1991.
The preliminary finding that nonprotein additions to the protein product of the ice-nucleating gene of Pseudomonas syringae or Erwinia herbicola are essential for ice nucleation at the warmest temperatures has led to experiments aimed at identifying possible linkages between the ice protein and the other components. It appears that the protein is coupled to various sugars through N- and O-glycan linkages. Mannose residues are apparently bound via an N-glycan bond to the amide nitrogen of one or more of the three essential asparagine residues in the unique amino-terminal portion of the protein. In turn, these mannose residues are involved in the subsequent attachment of phosphatidylinositol to the nucleation structure. This phosphatidylinositol-mannose-protein structure is the critical element in the class A nucleating structure. In addition to sugars attached to the asparagine residues, additional sugar residues appear to be attached by O-glycan linkages to serine and threonine residues in the primary repeating octapeptide, which makes up 70% of the total ice protein. These additional sugar residues include galactose and glucosamine and most likely additional mannose residues. These conclusions were based on (i) the changes in ice-nucleating activity due to the action of N- and O-glycanases, alpha- and beta-mannosidoses, and beta-galactosidase; (ii) immunoblot analyses of ice proteins in cell extracts after enzyme treatments; and (iii) the properties of transformed Ice+ Escherichia coli cells containing plasmids with defined amino-terminal and carboxyl-terminal deletions in the ice gene. Finally, evidence is presented that these sugar residues may play a role in aggregating the ice gene lipoglycoprotein compound into larger aggregates, which are the most effective ice nucleation structures.
初步研究发现，丁香假单胞菌或草生欧文氏菌冰核基因的蛋白质产物中添加非蛋白质成分对于在最温暖温度下的冰核形成至关重要，这引发了旨在确定冰蛋白与其他成分之间可能联系的实验。看来该蛋白质通过N - 和O - 聚糖键与各种糖类相连。甘露糖残基显然通过N - 聚糖键与蛋白质独特氨基末端部分三个必需天冬酰胺残基中一个或多个的酰胺氮相连。反过来，这些甘露糖残基参与了磷脂酰肌醇随后与成核结构的连接。这种磷脂酰肌醇 - 甘露糖 - 蛋白质结构是A类成核结构中的关键要素。除了与天冬酰胺残基相连的糖类外，额外的糖残基似乎通过O - 聚糖键与初级重复八肽中的丝氨酸和苏氨酸残基相连，该八肽占冰蛋白总量的70％。这些额外的糖残基包括半乳糖和葡糖胺，很可能还有额外的甘露糖残基。这些结论基于：（i）由于N - 和O - 聚糖酶、α - 和β - 甘露糖苷酶以及β - 半乳糖苷酶的作用导致的冰核活性变化；（ii）酶处理后细胞提取物中冰蛋白的免疫印迹分析；以及（iii）含有在冰基因中具有确定的氨基末端和羧基末端缺失的质粒的转化冰阳性大肠杆菌细胞的特性。最后，有证据表明这些糖残基可能在将冰基因脂糖蛋白化合物聚集成更大的聚集体中起作用，而这些聚集体是最有效的冰核结构。
