Krueger R C, Fields T A, Mensch J R, Schwartz N B
Department of Pediatrics, University of Chicago, Illinois 60637.
J Biol Chem. 1990 Jul 15;265(20):12088-97.
A lambda gt11 expression library containing cDNA from total chick embryo was screened with S103L, a rat monoclonal antibody which reacts specifically with the core protein of the chick cartilage chondroitin sulfate proteoglycan. One clone was identified which produced a 220-kDa beta-galactosidase/S103L-binding fusion protein. Sequencing the entire 1.5-kilobase cDNA insert showed that it contained a single open reading frame, which encoded a portion of the proteoglycan core protein from the chondroitin sulfate domain. This was confirmed by comparison with amino acid sequence data from peptide CS-B, which was derived from the chondroitin sulfate domain (Krueger, R.C., Jr., Fields, T. A., Hildreth, J., IV, and Schwartz, N.B. (1990) J. Biol. Chem. 265, 12075-12087). Furthermore, the 3' end of the insert overlapped with 23 bases at the 5' end of the published sequence for the C-terminal globular domain (Sai, S., Tanaka, T., Kosher, R. A., and Tanzer, M. L. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 5081-5085), which oriented this clone, as well as the CS peptide, along the protein core. The cDNA insert hybridized with a 9-kilobase mRNA from sternal chondrocytes as well as a similar sized message in brain but did not hybridize to any message from rat chondrosarcoma or from undifferentiated limb bud mesenchyme. In further studies, the fusion protein as well as a cyanogen bromide fragment (70 kDa) derived from it were isolated and shown to react with S103L, indicating that cleavage at methionine residues does not disrupt the antibody recognition site. Purification and N-terminal sequencing of the antigenic CNBr fragment derived from the fusion protein revealed that its N terminus is preceded by a methionine in the fusion protein and overlaps with the N terminus of peptide CS-B. As peptide CS-B is not recognized by S103L and the C terminus of peptide CS-B lies beyond the proteoglycan portion of the antigenic CNBr fragment, the S103L epitope is either contained within the 11 amino acids preceding the N terminus of peptide CS-B or it spans the clostripain cleavage site at the origin of the N terminus of peptide CS-B.
用S103L筛选了一个包含鸡胚总cDNA的λgt11表达文库，S103L是一种大鼠单克隆抗体，它能与鸡软骨硫酸软骨素蛋白聚糖的核心蛋白特异性反应。鉴定出一个克隆，它产生了一种220 kDa的β-半乳糖苷酶/S103L结合融合蛋白。对整个1.5千碱基的cDNA插入片段进行测序表明，它包含一个单一的开放阅读框，该阅读框编码了硫酸软骨素结构域中蛋白聚糖核心蛋白的一部分。通过与源自硫酸软骨素结构域的肽CS-B的氨基酸序列数据进行比较，证实了这一点（Krueger, R.C., Jr., Fields, T. A., Hildreth, J., IV, and Schwartz, N.B. (1990) J. Biol. Chem. 265, 12075 - 12087）。此外，插入片段的3'端与已发表的C端球状结构域序列（Sai, S., Tanaka, T., Kosher, R. A., and Tanzer, M. L. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 5081 - 5085）的5'端的23个碱基重叠，这确定了该克隆以及CS肽在蛋白核心上的方向。cDNA插入片段与胸骨软骨细胞的9千碱基mRNA以及脑中类似大小的信使RNA杂交，但不与大鼠软骨肉瘤或未分化肢芽间充质的任何信使RNA杂交。在进一步的研究中，分离出融合蛋白以及由其衍生的溴化氰片段（70 kDa），并显示它们与S103L反应，表明在甲硫氨酸残基处的切割不会破坏抗体识别位点。对源自融合蛋白的抗原性溴化氰片段进行纯化和N端测序表明，其N端在融合蛋白中前面有一个甲硫氨酸，并且与肽CS-B的N端重叠。由于肽CS-B不被S103L识别，并且肽CS-B的C端位于抗原性溴化氰片段的蛋白聚糖部分之外，因此S103L表位要么包含在肽CS-B的N端之前的11个氨基酸内，要么跨越肽CS-B的N端起始处的梭菌蛋白酶切割位点。
