Zhu A, Wang Z K, Beavis R
Lindsley F. Kimball Research Institute, The New York Blood Center, New York, New York 10021, USA.
Arch Biochem Biophys. 1998 Apr 1;352(1):1-8. doi: 10.1006/abbi.1998.0575.
alpha-N-Acetylgalactosaminidase (alphaNAGAL, EC 3.2.1.49) is an exoglycosidase specific for the hydrolysis of terminal alpha-linked N-acetylgalactosamine from oligosaccharide chains. After cloning of its cDNA, the recombinant alphaNAGAL (ralphaNAGAL) was produced in Pichia pastoris, a methylotrophic yeast strain. The enzyme was hyperglycosylated by the host cells, resulting in a protein with a molecular mass of approximately 50 kDa, which was 7 kDa larger than that of its native counterpart. When deglycosylated with endoglycosidase H under nondenaturing conditions, ralphaNAGAL remained fully active, suggesting that the glycosylation is not required for enzyme activity. Data derived from mass spectrometry indicated that all three putative N-glycosylation sites [Asn residues at positions 161 (N1), 185 (N2), and 369 (N3)] in the enzyme were glycosylated, and a high-mannose structure, which was possibly phosphorylated, was attached to the sites N1 and N2. In order to examine the effect of individual N-linked oligosaccharide chains on the expression of ralphaNAGAL in P. pastoris, we mutated each of the N-glycosylation sites, as well as all three sites in the same protein molecule, by substituting the Asn with a Gln residue. The results indicate that ralphaNAGAL mutations in any of the three glycosylation sites, N2 being the most profound, impaired the expression level, altered subcellular distribution, and decreased the efficiency of secretion. Our data suggest that the N-glycosylation of ralphaNAGAL expressed in P. pastoris may be important in protein folding and resistance to protease degradation during protein synthesis, although it is apparently not required for enzyme activity.
α-N-乙酰半乳糖胺酶（αNAGAL，EC 3.2.1.49）是一种外切糖苷酶，特异性地从寡糖链中水解末端α-连接的N-乙酰半乳糖胺。其cDNA克隆后，重组αNAGAL（ralphaNAGAL）在甲基营养酵母菌株毕赤酵母中产生。该酶被宿主细胞高度糖基化，产生一种分子量约为50 kDa的蛋白质，比其天然对应物大7 kDa。在非变性条件下用内切糖苷酶H去糖基化时，ralphaNAGAL仍保持完全活性，表明糖基化对酶活性不是必需的。质谱数据表明，该酶中所有三个假定的N-糖基化位点[161位（N1）、185位（N2）和369位（N3）的天冬酰胺残基]均被糖基化，并且一种可能被磷酸化的高甘露糖结构连接到N1和N2位点。为了研究单个N-连接寡糖链对ralphaNAGAL在毕赤酵母中表达的影响，我们通过将天冬酰胺替换为谷氨酰胺残基，对每个N-糖基化位点以及同一蛋白质分子中的所有三个位点进行了突变。结果表明，三个糖基化位点中任何一个发生ralphaNAGAL突变，其中N2的影响最为显著，都会损害表达水平、改变亚细胞分布并降低分泌效率。我们的数据表明，在毕赤酵母中表达的ralphaNAGAL的N-糖基化在蛋白质合成过程中的蛋白质折叠和抗蛋白酶降解方面可能很重要，尽管它显然对酶活性不是必需的。
