Chapman R L, Kane S E, Erickson A H
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
J Biol Chem. 1997 Mar 28;272(13):8808-16. doi: 10.1074/jbc.272.13.8808.
A single point mutation in the lysosomal proenzyme receptor-inhibiting sequence near the N terminus of mouse procathepsin L can result in glycosylation of a normally cryptic site near its C terminus. When alanine replaced His36, Arg38, or Tyr40, the nascent chain of the mutant protein cotranslationally acquired a high mannose oligosaccharide chain at Asn268. In contrast, when alanine replaced Ser34, Arg37, or Leu39, this second carbohydrate chain was not added. This alternating pattern of abnormal glycosylation suggested that propeptide residues 36-40 normally assume an extended conformation having the side chains of residues 36, 38, and 40 facing in the same direction. When tyrosine conservatively replaced His36 or lysine replaced Arg38, Asn268 was not glycosylated. But the procathepsin L mutant having phenylalanine in place of Tyr40 was glycosylated at Asn268, which indicates that the hydrogen bond between the hydroxyl group of Tyr40 and the carboxylate group of Asp82 is necessary for normal folding of the nascent proenzyme chain. Mutation of the adjacent alpha2p (ERININ) helix of the propeptide or addition of a C-terminal epitope tag sequence to procathepsin L also induced misfolding of the proenzyme, as indicated by addition of the second oligosaccharide chain. In contrast, the propeptide mutation KAKK99-102AAAA had no effect on carbohydrate modification even though it reduced the positive charge of the proenzyme. Misfolded mutant mouse procathepsin L was not efficiently targeted to lysosomes on expression in human HeLa cells, even though it acquired phosphate on mannose residues. The majority of the mutant protein was secreted after undergoing modification with complex sugars. Similarly, epitope-tagged mouse procathepsin L was not targeted to lysosomes in homologous mouse cells but was efficiently secreted. Since production of mature endogenous protease was not reduced in cells expressing the tagged protein, the tagged protein did not compete with endogenous procathepsin L for targeting to lysosomes.
小鼠组织蛋白酶L原N端附近的溶酶体酶原受体抑制序列中的单点突变，可导致其C端附近一个正常隐蔽位点的糖基化。当丙氨酸取代组氨酸36、精氨酸38或酪氨酸40时，突变蛋白的新生链在共翻译时在天冬酰胺268处获得一条高甘露糖寡糖链。相反，当丙氨酸取代丝氨酸34、精氨酸37或亮氨酸39时，第二条碳水化合物链未被添加。这种异常糖基化的交替模式表明，酶原肽残基36 - 40通常呈伸展构象，残基36、38和40的侧链朝向同一方向。当酪氨酸保守取代组氨酸36或赖氨酸取代精氨酸38时，天冬酰胺268未发生糖基化。但是用苯丙氨酸取代酪氨酸40的组织蛋白酶L原突变体在天冬酰胺268处发生了糖基化，这表明酪氨酸40的羟基与天冬氨酸82的羧基之间的氢键对于新生酶原链的正常折叠是必需的。酶原肽相邻的α2p（ERININ）螺旋的突变或在组织蛋白酶L原上添加C端表位标签序列也会诱导酶原错误折叠，这可通过添加第二条寡糖链来表明。相反，酶原肽突变KAKK99 - 102AAAA对碳水化合物修饰没有影响，尽管它降低了酶原的正电荷。错误折叠的突变小鼠组织蛋白酶L原在人HeLa细胞中表达时不能有效地靶向溶酶体，尽管它在甘露糖残基上获得了磷酸。大多数突变蛋白在经过复合糖修饰后被分泌。同样，表位标签化的小鼠组织蛋白酶L原在同源小鼠细胞中不能靶向溶酶体，但能有效地分泌。由于在表达标签化蛋白的细胞中成熟内源性蛋白酶的产生没有减少，标签化蛋白不会与内源性组织蛋白酶L原竞争靶向溶酶体。
