Expression and characterization of recombinant osteopontin peptides representing matrix metalloproteinase proteolytic fragments
Expression and characterization of recombinant osteopontin peptides representing matrix metalloproteinase proteolytic fragments
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DOI：
10.1016/j.matbio.2004.09.003
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发表时间：
2004-11-01
期刊：
影响因子：
6.9
通讯作者：
Liaw, L
中科院分区：
文献类型：
--
作者：
Gao, YA;Agnihotri, R;Liaw, L
骨桥蛋白（OPN）是一种分泌的、含有精氨酸-甘氨酸-天冬氨酸（RGD）的磷蛋白，由基质金属蛋白酶（MMP）家族成员进行蛋白水解修饰。我们先前定义了骨桥蛋白中的MMP-3和MMP-7切割位点，发现与全长骨桥蛋白相比，MMP切割片段的粘附和迁移活性增加。在本研究中，我们进行了突变分析的重组全长骨桥蛋白和产生的重组骨桥蛋白片段对应的MMP切割片段，其具有表观分子量为40，32，和25 kD的SDS-PAGE。L-167和L-211中的单残基突变不会消除MMP切割，尽管推定的C-末端片段的加工似乎受到L-167至（167）A突变的影响。尽管观察到全长OPN在可溶相与内皮细胞表面的结合更强，但与全长OPN相比，N-末端40-kD片段是更强的粘附底物。虽然32-kD片段显示出显着的结合内皮细胞表面，C-末端25-kD片段不与细胞表面相互作用。我们的数据表明，MMP切割的OPN的粘附活性增加是由N端40-kD片段负责。我们进一步分析了受体结合，使用竞争肽代表的α 4 β 1和α 9 β 1结合位点的40 kD的N-末端片段。使用Jurkat细胞，我们发现对应于（131）ELVTDFPTDLPATE（144）的肽对细胞粘附没有影响，而肽SVVYGLR竞争性抑制细胞粘附。这些162 166结果表明，在MMP切割的OPN中发现的较短基序（162）SVVYG（166）足以介导Jurkat细胞与受体的细胞粘附，包括β 1整联蛋白，其先前已被表征为结合SVVYGLR序列。(C)2004年Elsevier B. V./国际矩阵生物学学会。All rights reserved.
Osteopontin (OPN) is a secreted, arginine-glycine-aspartic acid (RGD)-containing phosphoprotein proteolytically modified by members of the matrix metalloproteinase (MMP) family. We previously defined the MMP-3 and MMP-7 cleavage sites in OPN and found increased adhesive and migratory activity of a pool of MMP-cleaved fragments compared to full-length OPN. In the present study, we performed mutational analysis of recombinant full-length OPN and generated recombinant OPN fragments corresponding to the MMP-cleaved fragments, which have apparent molecular weights of 40, 32, and 25 kD by SDS-PAGE. Single residue mutations in L-167 and L-211 do not abrogate MMP cleavage although processing of the putative C-terminal fragment appears to be affected by a L-167 to (167)A mutation. The N-terminal 40-kD fragment was a stronger adhesive substrate compared to full-length OPN despite the observation that full-length OPN displayed greater binding in soluble phase to endothelial cell surfaces. While the 32-kD fragment showed significant binding to endothelial cell surfaces, the C-terminal 25-kD fragment did not interact with cell surface. Our data indicate that the increased adhesive activity of MMP-cleaved OPN was accountable by the N-terminal 40-kD fragment. We further analyzed receptor binding, using competition with peptides representing the alpha4beta1 and alpha9beta1 binding sites in the 40-kD N-terminal fragment. Using Jurkat cells, we found that a peptide corresponding to (131)ELVTDFPTDLPATE(144) had no effect on cell adhesion, whereas the peptide SVVYGLR competitively inhibited cell adhesion. These 162 166 results suggest that a shorter motif that is found in MMP-cleaved OPN, (162)SVVYG(166), is sufficient to mediate cell adhesion of Jurkat cells to receptors, including the betal integrins, which have been previously characterized to bind the SVVYGLR sequence. (C) 2004 Elsevier B.V./International Society of Matrix Biology. All rights reserved.