New monoclonal antibody promotes research of cartilage metabolism

The expression mechanism of SK-28 positive, a keratan sulfate-bearing species of aggrecan, may correlate with the maturation of articular cartilage in vivo.

Issue: Issue 5 2004

ByKatsuji Shimizu, MD, DMSc

Katsuji Shimizu, MD, DMSc [photo] --- Katsuji Shimizu, MD, DMSc

Aggrecan is the major space-filling proteoglycan in articular cartilage that provides the tissue with mechanical properties of reversible compressibility. In joint pathology such as that seen in osteoarthritis and rheumatoid arthritis, aggrecan loss leads to progressive cartilage degradation. Those enzymes responsible for cartilage and collagen degradation are called matrix proteinases because their chief site of action is in the extracellular matrix.

Aggrecanase and matrix-metalloproteinase (MMP) are well-characterized matrix proteinases responsible for the degradation of aggrecan. However, since aggrecanase and MMPs alone cannot explain every event of aggrecan degradation, we believe that other types of matrix proteinases exist.

Calpain is a neutral cystein proteinase that researchers first discovered in the rat brain. It has long been considered an intracellular proteinase. Recent studies revealed that calpain is present in the extracellular matrix during biological events such as endochondral ossification in growth cartilage and fracture healing, osteoarthritis and rheumatoid arthritis.

Calpain itself has unique regulatory activity on aggrecan. It cleaves aggrecan from the C-terminal to the N-terminal, preserves the domain structures of aggrecan after digestion, and may serve to switch between the maximal and limited structure/function of aggrecan. However, researchers have not demonstrated that calpain-mediated bioactive fragments, or species, of aggrecan exist in vivo until the development of our new antibody.

A new antibody shows promise

We recently produced a new monoclonal antibody (SK-28) in the department of orthopaedic surgery at Gifu University to recognize the C-terminal neoepitope at M710VTQVGPGVA719. It detects a novel cleavage site of aggrecan, which is specific to calpain and distinct from aggrecanase and MMPs. In studies using SK-28, we found a high prevalence of cleavage products present as the major G1-G2 and keratan sulfate (KS)-bearing large aggrecan species.

The species appear within mature bovine articular cartilage extracts and are responsible for maintaining aggregating capacity during the course of isolation within A₁A₁ subfragments. The expression mechanism of these or the KS-bearing species of aggrecan appear to be directly correlated with the maturation/aging mechanism of articular cartilage in vivo.

SK-28 will be a useful tool to study in this application as well as in many unexplained physiological and pathological events in aggrecan metabolism.

Katsuji Shimizu, MD, DMSc, is professor and chairman of the department of orthopaedic surgery at Gifu University School of Medicine, Gifu, Japan. He is an editor of Orthopaedics Today.

For your information:

Oshita H, Sandy JD, Suzuki K, et al. Mature bovine articular cartilage contains abundant aggrecan that is C-terminally truncated at Ala(719)-Ala(720), a site which is readily cleaved by m-calpain. Biochem J. 2004; 382:1253-9.