Vijayasaradhi S, Doskoch P M, Houghton A N
Memorial Sloan-Kettering Cancer Center, New York, New York 10021.
Exp Cell Res. 1991 Oct;196(2):233-40. doi: 10.1016/0014-4827(91)90256-t.
A 75-kDa melanosomal glycoprotein (gp75) is the product of a gene that maps to the b (brown) locus, a genetic locus that determines coat color in the mouse. The b locus is conserved (88% identity) between mouse and human. The mouse monoclonal antibody TA99 was used to study the biosynthesis and processing of gp75. gp75 was synthesized as a 55-kDa polypeptide, glycosylated by addition and processing of five or more Asn-linked carbohydrate chains through the cis and trans Golgi, and transported to melanosomes as a mature 75-kDa form. Synthesis and processing of gp75 was rapid (T1/2 less than 30 min), and early steps in processing were required for efficient export of gp75 to melanosomes. Fully processed mature gp75 was quite stable (T1/2 = 22-24 h) in the melanosome. Digestion of high-mannose carbohydrate chains with endo-beta-N-acetylglucosaminidase H revealed two alternative processed forms of gp75 that differed in the number or composition of complex-type carbohydrate chains. The rate of synthesis and movement through intracellular membrane compartments was the same for both glycosylated forms. Studies with inhibitors of steps in oligosaccharide processing showed that alternative forms of gp75 were generated during trimming reactions by mannosidase IA/IB and that further maturation resulted in the two mature forms of gp75. We propose that the kinetics of biosynthesis and processing reflect events in the biogenesis and maturation of melanosomes.
一种75千道尔顿的黑素小体糖蛋白（gp75）是一个基因的产物，该基因定位于b（棕色）位点，这是一个决定小鼠毛色的遗传位点。b位点在小鼠和人类之间是保守的（88%同源）。小鼠单克隆抗体TA99被用于研究gp75的生物合成和加工过程。gp75最初合成时是一种55千道尔顿的多肽，通过顺式和反式高尔基体添加并加工五条或更多条天冬酰胺连接的碳水化合物链进行糖基化，然后以成熟的75千道尔顿形式转运到黑素小体。gp75的合成和加工速度很快（半衰期小于30分钟），加工的早期步骤是gp75有效输出到黑素小体所必需的。完全加工成熟的gp75在黑素小体中相当稳定（半衰期 = 22 - 24小时）。用内切β-N-乙酰葡糖胺糖苷酶H消化高甘露糖碳水化合物链后，发现了gp75的两种不同加工形式，它们在复合型碳水化合物链的数量或组成上有所不同。两种糖基化形式的合成速度和通过细胞内膜隔室的移动速度相同。对寡糖加工步骤抑制剂的研究表明，gp75的不同形式是在甘露糖苷酶IA/IB的修剪反应过程中产生的，进一步成熟导致了gp75的两种成熟形式。我们认为，生物合成和加工的动力学反映了黑素小体生物发生和成熟过程中的事件。
