Researchers identify proteins with strong ability to bind to lubricin

NEW ORLEANS — Type IV collagen and tenascin-C were among several proteins which showed a strong ability to bind to lubricin in a recent study presented here.

Carl R. Flannery, PhD, presented his findings at the 56th Annual Meeting of the Orthopaedic Research Society.

Though studies have shown that lubricin localizes to articular cartilage surfaces via mechanisms dependent on protein secondary structure, Flannery said that relatively little is known about the specific molecular interactions that facilitate the bonding.

“[We are] trying to understand a little more about the basic biology of lubricin and how it functions in the joint,” Flannery said during his presentation.

Immunoprecipitation

Flannery and his group extracted the surface layer of macroscopically normal human articular cartilage specimens with 4M guanidine-HCl and dialyzed them exhaustively against proteomics-based studies. Re-associated lubricin-ligand complexes were immunoprecipitated (IP) using an anti-lubricin monoclonal antibody (MAb) conjugated to MyOne Dynabeads [Invitrogen]. Control IP experiments were conducted using normal mouse immunoglobulin.

Commercially available proteins (collagens, tenascin-C, fibronectin, cartilage oligomeric matrix protein, vitronectin, decorin and bovine serum albumin) were used to coat microtiter plates. Titrated amounts of recombinant lubricin were added to the wells, and bound protein was detected using an anti-lubricin MAb, followed by spectrophotometric detection of colorimetric product.

Solid-phase binding assays

Flannery reported that more than three distinct peptides were present in the anti-lubricin IP samples, and the number of distinct peptides was enriched 2-fold or greater relative to the control samples. In addition to lubricin itself, type IV collagen, tenascin-C, aggrecan, fibronectin, cartilage oligomeric matrix protein, vitronectin and decorin were among the most abundant proteins identified. These components were then further tested in solid-phase binding assays.

Flannery noted that collagen triple helical, leucine-rich and thrombospondin-like repeats, as well as von Willebrand factor (vWF) type A, epidermal growth factor-like and fibronectin type III domains were among the most abundant in the study’s topical proteins, suggesting that the potential for multi-valent interactions involved in lubricin localization at cartilage surfaces.

Still, he said, there is a lot of work to be done.

“It is probably not as simple as single-molecule binding, but I think if we can start to unravel it, hopefully we will make some progress in further understanding how this very interesting molecule works within the joint,” Flannery concluded.

Reference:
Flannery CR, Yang Z, Zeng W, et al. Proteomic identification of novel lubricin-binding ligands at cartilage surfaces. Paper 164. Presented at the 56th Annual Meeting of the Orthopaedic Research Society. March 6-9, 2010. New Orleans.

It was a wonderful talk and it stimulated a number of thoughts. I got into this business not studying or utilizing a cartilage bearing, but using a bearing that oscillated latex on glass years ago. Occasionally I still use it in my laboratory. The lubricin purified from synovial fluid replicates the full lubricating characteristics of synovial fluid in that bearing system. That goes to show that the lubricating effect does not necessarily need biological surfaces – although I think in general it does … but this will lubricate non-biological surfaces as well.

– Gregory D. Jay, MD, PhD
Professor of Emergency Medicine and Engineering
Department of Emergency Medicine and Division of Engineering
Brown University

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