Phosphacan, a chondroitin sulfate proteoglycan of brain that interacts with neurons and neural cell-adhesion molecules, is an extracellular variant of a receptor-type protein tyrosine phosphatase.

We have identified cDNA clones encoding a chondroitin sulfate proteoglycan of rat brain (previously designated 3F8 and now named phosphacan) that binds to neurons and neural cell-adhesion molecules. A sequence of 1616 amino acids deduced from a 4.8-kb open reading frame contains the N-terminal amino acid sequence of the 3F8 core glycoprotein as well as four internal CNBr, tryptic, and endoproteinase Lys-C peptide sequences from the proteoglycan. The deduced amino acid sequence, beginning with a 24-amino acid signal peptide, reveals an N-terminal domain of 255 amino acids homologous to carbonic anhydrases. The entire amino acid sequence deduced from our cDNA clones corresponds to the extracellular portion of a human receptor-type protein tyrosine phosphatase (RPTP zeta/beta) with which it has 76% identity, and the proteoglycan may represent an mRNA splicing variant of the larger transmembrane protein. RNA analysis demonstrated that a probe to the N-terminal carbonic anhydrase domain of the proteoglycan hybridizes with rat brain mRNA of 9.5, 8.4, and 6.4 kb, whereas probes to the phosphatase domains hybridize with only the 9.5-kb message and with the 6.4-kb message (which corresponds to a previously identified variant of the transmembrane protein in which half of the extracellular domain is deleted). The 30 N-terminal amino acids of the 3H1 chondroitin/keratan sulfate proteoglycan of brain are identical to those of the 3F8 proteoglycan, and six internal tryptic peptide sequences also matched those found in sequenced peptides of the 3F8 proteoglycan and/or amino acid sequences deduced from the cDNA clones. We therefore conclude that the 3H1 chondroitin/keratan sulfate proteoglycan and the 3F8 chondroitin sulfate proteoglycan represent glycosylation and possible extracellular splicing variants of a receptor-type protein tyrosine phosphatase. These proteoglycans may modulate cell interactions and other developmental processes in nervous tissue through heterophilic binding to cell-surface and extracellular matrix molecules, and by competition with ligands of the transmembrane phosphatase. IMAGES:

本研究鉴定得到编码大鼠脑硫酸软骨素蛋白聚糖（此前命名为3F8，现命名为phosphacan）的互补DNA（complementary DNA, cDNA）克隆，该蛋白聚糖可与神经元及神经细胞黏附分子（neural cell-adhesion molecules）结合。由4.8 kb开放阅读框（open reading frame, ORF）推导得到的1616个氨基酸序列，既包含3F8核心糖蛋白的N端氨基酸序列，也包含该蛋白聚糖的4个内源性溴化氰（CNBr）、胰蛋白酶及内蛋白酶Lys-C（endoproteinase Lys-C）酶解肽段序列。该推导氨基酸序列以一段24个氨基酸的信号肽（signal peptide）起始，其255个氨基酸的N端结构域（N-terminal domain）与碳酸酐酶（carbonic anhydrases）具有同源性。本研究中cDNA克隆推导得到的完整氨基酸序列，与人受体型蛋白酪氨酸磷酸酶ζ/β（receptor-type protein tyrosine phosphatase ζ/β, RPTP ζ/β）的胞外结构域（extracellular portion）完全对应，二者序列一致性达76%；该蛋白聚糖可能属于该跨膜蛋白的一种mRNA剪接变体（mRNA splicing variant）。RNA分析（RNA analysis）结果显示，靶向该蛋白聚糖N端碳酸酐酶结构域的分子探针（probe），可与大鼠脑内9.5 kb、8.4 kb及6.4 kb的mRNA杂交；而靶向磷酸酶结构域的分子探针，仅能与9.5 kb和6.4 kb的转录本杂交（其中6.4 kb转录本对应此前已鉴定的跨膜蛋白剪接变体，该变体缺失了一半的胞外结构域）。脑内3H1型硫酸软骨素/硫酸角质素蛋白聚糖的30个N端氨基酸与3F8型蛋白聚糖完全一致，且6个内源性胰蛋白酶酶解肽段序列，也与3F8型蛋白聚糖的测序肽段及/或本研究cDNA克隆推导得到的氨基酸序列相匹配。综上，我们认为3H1型硫酸软骨素/硫酸角质素蛋白聚糖与3F8型硫酸软骨素蛋白聚糖，属于同一受体型蛋白酪氨酸磷酸酶的糖基化（glycosylation）变体及可能的胞外剪接变体。这类蛋白聚糖可通过与细胞表面及细胞外基质（extracellular matrix）分子发生异嗜性结合（heterophilic binding），以及竞争性结合该跨膜磷酸酶的配体，来调控神经组织中的细胞相互作用及其他发育过程。图像：