Characterization of recombinant amino-terminal NC4 domain of human collagen IX: interaction with glycosaminoglycans and cartilage oligomeric matrix protein.

Tero Pihlajamaa,Frank Zaucke,Ilkka Kilpelaïnen,Johan Peränen,Joni Ylöstalo,Leena Valmu,Hilkka Lankinen,Luitgard Spitznagel,Juha Jäälinoja,Anne Puustinen,Leena Ala-Kokko,Matthias Mörgelin,Patrik Maurer,Silke Gösling

doi:10.1074/jbc.m402865200

Abstract

The N-terminal NC4 domain of collagen IX is a globular structure projecting away from the surface of the cartilage collagen fibril. Several interactions have been suggested for this domain, reflecting its location and its characteristic high isoelectric point. In an attempt to characterize the NC4 domain in more detail, we set up a prokaryotic expression system to produce the domain. The purified 27.5-kDa product was analyzed for its glycosaminoglycan-binding potential by surface plasmon resonance and solid-state assays. The results show that the NC4 domain of collagen IX specifically binds heparin with a K(d) of 0.6 microm, and the full-length recombinant collagen IX has an even stronger interaction with heparin, with an apparent K(d) of 3.6 nm. The heparin-binding site of the NC4 domain was located in the extreme N terminus, containing a heparin-binding consensus sequence, whereas electron microscopy suggested the presence of at least three additional heparin-binding sites on full-length collagen IX. The NC4 domain was also shown to bind cartilage oligomeric matrix protein. This interaction and the association of cartilage oligomeric matrix protein with other regions of collagen IX were found to be heparin-competitive. Circular dichroism analyses of the NC4 domain indicated the presence of stabilizing disulfide bonds and a thermal denaturation point of about 80 degrees C. The pattern of disulfide bond formation within the NC4 domain was identified by tryptic peptide mass mapping of the NC4 in native and reduced states. A similar pattern was demonstrated for the NC4 domain of full-length recombinant collagen IX.

Highlights

The N-terminal NC4 domain of collagen IX is a globular structure projecting away from the surface of the cartilage collagen fibril
The NC4 domain of collagen IX is seen in electron microscopy as a compact globulus projecting away from the fibril body, with the COL3 domain acting as a spacer arm [6, 7]
Analysis of Disulfide Bonding Pattern—In order to isolate the NC4 domain from full-length collagen molecules, recombinant human collagen IX (rcIX) was digested with collagenase (Sigma), the reaction mixture was passed through a heparin-Sepharose affinity column (Amersham Biosciences), and the bound material was eluted with an increasing salt gradient and analyzed by SDS-PAGE

Summary

INTERACTION WITH GLYCOSAMINOGLYCANS AND CARTILAGE OLIGOMERIC MATRIX PROTEIN*

The NC4 domain of collagen IX is seen in electron microscopy as a compact globulus projecting away from the fibril body, with the COL3 domain acting as a spacer arm [6, 7]. This location and the high theoretical pI of the NC4 domain implicate collagen IX as a potential docking molecule, possibly connecting the host fibril to adjacent collagen fibrils or to other macromolecules of the extracellular matrix [8]. No research has yet been reported, on the glycosaminoglycan binding properties of the NC4 domain or full-length collagen IX

Studies in vitro have demonstrated that cartilage oligomeric

EXPERIMENTAL PROCEDURES

RESULTS

Measured mass

DISCUSSION