Aggrecan is a large aggregating proteoglycan of articular cartilage. It is also found in aorta, discs and tendons (1,2). The aggrecan core protein consists of 2317aa (2). Up to 130 glucosaminoglycan chains are attached to the core protein. The total molecular mass can reach 2.2-3 x 10e3kD (4). Within the aggrecan molecule, 3 global domains G1, G2 and G3 can be distinguished. Domains G1 and G2 are connected by a rod-shaped polypeptide called interglobular domain (IGD). The sequence between domains G2 and G3 contains attachment regions for keratan sulfate and chondroitin sulfate chains. Aggrecan interacts via the G1domain with hyaluronan and link protein to form large aggregates. Such aggregates can contain up to 50-100 aggrecan monomers noncovalently bound to a single hyaluronan chain through 2 link proteins (1,2,4). The aggregates form a hydrated gel-like structurem, which endowes cartilage with resistibility to compression and deformation. Degradation of aggrecan appears to initiate at the C-terminus. The population of aggrecan molecules without the G3 domain increases with aging (5). Isolated aggrecanases cleave aggrecan at 4 sites within the chrondroitin sulfate-rich region (sites E1667-G1668, E1480-G1481, E1771-A1772, E1871-L1872) and 1 site within the interglobular domain (E373-A374) (6). Cleavage at the latter site had been documented by analysis of cartilage proteoglycan breakdown products in rheumatoid and osteoarthritis (7).
To measure aggrecanase activity, an artificial recombinant protein composed of aggrecan interglobular domain with flanking FLAG-sequence and human immunoglobulin G1 constant region was first used by Hughes et al. (8).
Source:The polypeptide connecting human aggrecan globular domains 1 and 2 (T331-G458) is expressed in E. coli with a C-terminal His-tag. The recombinant protein contains cleavage sites for aggrecanases (E373-A374) and matrix metalloproteinases (N341-F342). It comprises the following amino acids: TAEDFVDIPENFFGVGGEEDITVQTVWPDMELPLPRNITEGEARGSVILTVKPIFEVSPSPLEPEEPFTFAPEIGATAFAEVENETGEATRPWGFPTPGLGPATAFTSEDLVVQVTAVPGQPHLPGG(His-tag). The calculated Mr of the His-tagged protein is 15.493kD.
Applications:Aggrecan interglobular domain is used as substrate for aggrecanases and matrix metalloproteinases. For proteinase activity measurements, the protein is incubated with proteinase for various time intervals. Thereafter, aliquots of the incubation mixture are analyzed by SDS-PAGE or by ELISA. Upon cleavage with aggrecanases the apparent Mr of aggrecan interlobular domain in SDS-PAGE is reduced from 21kD to ~13kD. Quantitative measurement of aggrecanase cleavage requires a neoepitope antibody with specificity for the N-terminus ARGSVILT... appearing upon hydrolysis. The fragment with the newly formed N-terminus is fixed by the neoepitope antibody to a microplate and quantified with an anti-His-tag antibody. In analogy, cleavage by matrix metalloproteinases can be measured with antibodies to neoepitopes appearing upon action of these enzymes.
Storage and Stability:For long-term storage, aliquot to avoid repeated freezing and thawing and freeze at -70°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Aliquots are stable for 6 months.
仕様
Size：50ug
宿主：
Source Antigen：Recombinant, E. coli
Grade：Highly Purified
Purity：Recombinant aggrecan interglobular domain appears as a major band at about 21kD in SDS-PAGE, representing ≥ 90% of total protein in the preparation.
Form：Supplied as a liquid in 50mM Tris HCI, pH 7.5, 150mM sodium chloride, 5mM calcium chloride.
