Identification of Hyaluronan-binding Domains of Aggrecan*
- From the ‡Craniofacial Developmental Biology and Regeneration Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892-4370, the§Laboratory of Hemostasis, Division of Hematology, Center for Biologics Evaluation and Research, U. S. Food and Drug Administration, Rockville, Maryland 20852, and the ¶Institute for Molecular Science of Medicine, Aichi Medical University, Nagakute, Aichi, 480-11, Japan
Abstract
Aggrecan, a large cartilage proteoglycan, interacts with hyaluronan (HA), to form aggregates which function to resist compression in joints. The N-terminal region of aggrecan contains two structurally related globular domains, G1 and G2 separated by IGD domain. The G1 domain consists of three subdomains, A, B, and B′, structural features characteristic to many other HA-binding proteoglycans. Here, we studied the interaction of aggrecan domains with HA using recombinant proteins expressed in 293 cells, an embryonal kidney cell line. Deglycosylation of the recombinant aggrecan fragment reduced the HA binding activity. We found that both the B and B′ subdomains were required for HA binding and that a single module of A, B, or B′ was unable to bind HA. The A subdomain increased the HA binding activity of the B-B′ region. The G2 domain had no HA binding activity confirming previous reports. Studies of HA-binding properties using a BIAcoreTM biosensor system revealed that the K D of recombinant aggrecan fragment (AgW) consisting of G1, IGD, and G2 was 0.226 μm, whereas the K D of another HA-binding protein, native bovine link protein, is 0.089 μm. In contrast, AgMut11 which lacked subdomain A showed little HA binding activity. AgMut12 consisting of only B-B′ had a 3.4-fold lower affinity and AgMut13 containing A-B-B′ was 1.5-fold lower than AgW. These results suggest that carbohydrates are essential for high level aggrecan binding to HA and that the A subdomain of aggrecan functions in a cooperative manner with subdomains B and B′.
Footnotes
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↵* This work was supported in part by the Seikagaku Corp.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‖ Visiting Scholar in residence at the Fogarty International Center, National Institutes of Health.
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↵** To whom correspondence should be addressed: Bldg. 30, Rm. 405, National Institute of Dental Research, National Institutes of Health, 30 Convent Dr. MSC 4370, Bethesda, MD 20892-4370. Tel.: 301-496-2111; Fax: 301-402-0897; E-mail: yamada{at}yoda.nidr.nih.gov.
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↵1 The abbreviations used are: HA, hyaluronan; CPC, cetylpyridinium chloride; HABR, hyaluronan-binding region; IGD, interglobular domain; PAGE, polyacrylamide gel electrophoresis; TFMSA, trifluoromethane sulfonic acid; TSG-6, tumor necrosis factor α-stimulated gene-6; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; PE, phosphatidylethanolamine.
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↵2 Y. Yamada, unpublished data.
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- Received June 13, 1997.
- Revision received August 13, 1997.
