Kaetzel D M, Morgan D, Reid J D, Fenstermaker R A
Department of Pharmacology, College of Medicine, Chandler Medical Center, University of Kentucky, Lexington 40536, USA.
Biochim Biophys Acta. 1996 Dec 5;1298(2):250-60. doi: 10.1016/s0167-4838(96)00136-7.
Pulse-chase analysis of human platelet-derived growth factor (PDGF) B-chain was conducted in stably transfected Chinese hamster ovary cells to determine precisely the kinetics of processing, intracellular trafficking and secretion. Newly synthesized 31 kDa monomers of the B-chain (p31) dimerized rapidly via disulfide bonds to a p54 species (t1/2 < 30 min). The p54 dimer was processed to a group of intracellular, cell surface (suramin-releasable) and secreted forms whose rates of appearance and disappearance from the cell were measured over a 48 h period. The newly synthesized p31 species was quantitatively converted to p27 by treatment with endoglycosidase H, consistent with efficient N-glycosylation at a site in the N-terminal propeptide region (Asn63-Met64-Thr65). Interruption of B-chain glycosylation by oligodeoxynucleotide-directed mutagenesis resulted in a significant increase in suramin-releasable forms at the cell surface (p34-38) and a concomitant decrease in accumulation of an intracellular p24 species. The glycosylation-defective mutant exhibited slight increases in receptor binding and mitogenic activity. Our results suggest that N-linked glycosylation of the B-chain is not important for formation of mitogenically active protein, but that it plays a role in early intracellular sorting and proteolytic processing events.
在稳定转染的中国仓鼠卵巢细胞中对人血小板衍生生长因子（PDGF）B链进行脉冲追踪分析，以精确确定加工、细胞内运输和分泌的动力学。新合成的31 kDa B链单体（p31）通过二硫键迅速二聚化为p54形式（半衰期<30分钟）。在48小时内测量了p54二聚体加工成一组细胞内、细胞表面（可被苏拉明释放）和分泌形式的出现和消失速率。用内切糖苷酶H处理后，新合成的p31形式定量转化为p27，这与在N端前肽区域（Asn63-Met64-Thr65）的一个位点上有效的N-糖基化一致。通过寡脱氧核苷酸定向诱变中断B链糖基化导致细胞表面可被苏拉明释放的形式（p34-38）显著增加，同时细胞内p24形式的积累减少。糖基化缺陷突变体在受体结合和促有丝分裂活性方面略有增加。我们的结果表明，B链的N-连接糖基化对有丝分裂活性蛋白的形成并不重要，但它在早期细胞内分选和蛋白水解加工事件中起作用。
