Not All Lubricin Isoforms Are Substituted with a Glycosaminoglycan Chain

Abstract

Lubricin, also referred to as superficial zone protein, has been reported to be a proteoglycan. However, the structure of its glycosaminoglycan chain has not been well characterized, and this study was undertaken to investigate the structure of the glycosaminoglycan chain that decorated lubricin in human synovial fluid to provide insight into its biological role. Lubricin was detected as a major band at approximately 360 kDa which co-migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a chondroitin sulfate (CS)-containing proteoglycan that was detected by both monoclonal antibodies (MAb) 2-B-6 and MAb 3-B-3 after chondroitinase ABC treatment and keratan sulfate (KS) that was detected by MAb 5-D-4. Further analysis of lubricin-containing fractions that eluted from an anion exchange column indicated that the major population of lubricin could be separated from the CS and KS stubs which indicated that this fraction of lubricin was not decorated with glycosaminoglycan chain and was the glycoprotein form of lubricin. Lubricin present in fractions that also contained CS was found to be decorated with CS structures which were reactive with MAb 3-B-3 after chondroitinase ABC digestion using a sandwich enzyme-linked immunosorbent assay approach. Aggrecan was not found to form complexes with lubricin in synovial fluid which confirmed that the MAb 3-B-3 CS and MAb 5-D-4 KS structures decorated lubricin. These data demonstrate that lubricin present in human synovial fluid was a heterogeneous population with both glycoprotein and proteoglycan forms.

• lubricin
• chondroitin sulfate
• keratan sulfate
• proteoglycan
• synovial fluid

ACKNOWLEDGMENTS

We acknowledge Prof. Bruce Caterson, Cardiff University, for the supply of the KS (clone 5-D-4), KS stubs (clone BKS-1), and CS linkage region (clones 1-B-5, 2-B-6, and 3-B-3) antibodies. The monoclonal antibody clone 12C5 was developed by Prof. Richard Asher and obtained from the Developmental Studies, Hybridoma Bank, developed under the auspices of the National Institute of Child Health and Human Development and maintained by the Department of Biology, The University of Iowa, Iowa City, IA 52242, USA. This work was supported by the Australian Research Council through the Linkage Project Grant Scheme (grant number LP0455407).

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.