Mercuri F A, Maciewicz R A, Tart J, Last K, Fosang A J
University of Melbourne, Department of Paediatrics, Orthopaedic Molecular Biology Research Unit and Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville, 3052, Australia.
J Biol Chem. 2000 Oct 20;275(42):33038-45. doi: 10.1074/jbc.275.42.33038.
We have expressed G1-G2 mutants with amino acid changes at the DIPEN(341) downward arrow(342)FFGVG and ITEGE(373) downward arrow(374)ARGSV cleavage sites, in order to investigate the relationship between matrix metalloproteinase (MMP) and aggrecanase activities in the interglobular domain (IGD) of aggrecan. The mutation DIPEN(341) to DIGSA(341) partially blocked cleavage by MMP-13 and MMP-8 at the MMP site, while the mutation (342)FFGVG to (342)GTRVG completely blocked cleavage at this site by MMP-1, -2, -3, -7, -8, -9, -13, -14. Each of the MMP cleavage site mutants, including a four-amino acid deletion mutant lacking residues ENFF(343), were efficiently cleaved by aggrecanase, suggesting that the primary sequence at the MMP site had no effect on aggrecanase activity in the IGD. The mutation (374)ARGSV to (374)NVYSV completely blocked cleavage at the aggrecanase site by aggrecanase, MMP-8 and atrolysin C but had no effect on the ability of MMP-8 and MMP-13 to cleave at the Asn(341) downward arrowPhe bond. Susceptibility to atrolysin C cleavage at the MMP site was conferred in the DIGSA(341) mutant but absent in the wild-type, (342)GTRVG, (374)NVYSV, and deletion mutants. To further explore the relationship between MMP and aggrecanase activities, sequential digest experiments were done in which MMP degradation products were subsequently digested with aggrecanase and vice versa. Aggrecanase-derived G1 domains with ITEGE(373) C termini were viable substrates for MMPs; however, MMP-derived G2 fragments were resistant to cleavage by aggrecanase. A 10-mer peptide FVDIPENFFG, which is a substrate analogue for the MMP cleavage site, inhibited aggrecanase cleavage at the Glu(373) downward arrowAla bond. This study demonstrates that MMPs and aggrecanase have unique substrate recognition in the IGD of aggrecan and suggests that sequences at the C terminus of the DIPEN(341) G1 domain may be important for regulating aggrecanase cleavage.
我们表达了在DIPEN(341)↓(342)FFGVG和ITEGE(373)↓(374)ARGSV裂解位点发生氨基酸变化的G1 - G2突变体，以研究基质金属蛋白酶（MMP）与聚集蛋白聚糖球间结构域（IGD）中聚集蛋白聚糖酶活性之间的关系。将DIPEN(341)突变为DIGSA(341)部分阻断了MMP - 13和MMP - 8在MMP位点的裂解，而将(342)FFGVG突变为(342)GTRVG则完全阻断了MMP - 1、-2、-3、-7、-8、-9、-13、-14在此位点的裂解。每个MMP裂解位点突变体，包括缺失ENFF(343)残基的四氨基酸缺失突变体，都能被聚集蛋白聚糖酶有效裂解，这表明MMP位点的一级序列对IGD中聚集蛋白聚糖酶的活性没有影响。将(374)ARGSV突变为(374)NVYSV完全阻断了聚集蛋白聚糖酶、MMP - 8和抗胰蛋白酶C在聚集蛋白聚糖酶位点的裂解，但对MMP - 8和MMP - 13裂解Asn(341)↓Phe键的能力没有影响。DIGSA(341)突变体赋予了MMP位点对抗胰蛋白酶C裂解的敏感性，而野生型、(342)GTRVG、(374)NVYSV和缺失突变体则没有。为了进一步探究MMP与聚集蛋白聚糖酶活性之间的关系，进行了顺序消化实验，即先用MMP消化产物随后再用聚集蛋白聚糖酶消化，反之亦然。具有ITEGE(373) C末端的聚集蛋白聚糖酶衍生的G1结构域是MMPs的可行底物；然而，MMP衍生的G2片段对聚集蛋白聚糖酶的裂解具有抗性。一种10肽FVDIPENFFG，它是MMP裂解位点的底物类似物，抑制了聚集蛋白聚糖酶在Glu(373)↓Ala键处的裂解。这项研究表明，MMPs和聚集蛋白聚糖酶在聚集蛋白聚糖的IGD中具有独特的底物识别能力，并表明DIPEN(341) G1结构域C末端的序列可能对调节聚集蛋白聚糖酶的裂解很重要。
