Consequences of Disease-causing Mutations on Lubricin Protein Synthesis, Secretion, and Post-translational Processing

David K Rhee,Jose Marcelino,Sulaiman Al-Mayouf,Deborah K Schelling,Cynthia F Bartels,Yajun Cui,Ronald Laxer,Raphaela Goldbach-Mansky,Matthew L Warman

doi:10.1074/jbc.m505401200

Abstract

Lubricin, a protein product of the gene PRG4, is a secreted mucin-like proteoglycan that is a major lubricant in articulating joints. Mutations in PRG4 cause the autosomal recessive, human disorder camptodactyly-arthropathy-coxa vara-pericarditis syndrome. We developed rabbit polyclonal antibodies against human lubricin to determine the consequence of disease-causing mutations at the protein level and to study the protein's normal post-translational processing. Antiserum generated against an epitope in the amino-terminal portion of lubricin detected protein in wild-type synovial fluid and in conditioned media from wild-type cultured synoviocytes. However, the antiserum did not detect lubricin in synovial fluid or cultured synoviocytes from several patients with frameshift or nonsense mutations in PRG4. Antiserum generated against an epitope in the protein's carboxyl-terminal, hemopexin-like domain identified a post-translational cleavage event in wild-type lubricin, mediated by a subtilisin-like proprotein convertase (SPC). Interestingly, in contrast to wild-type lubricin, one disease-causing mutation that removes the last 8 amino acids of the protein, including a conserved cysteine residue, was not cleaved within the hemopexin-like domain when expressed in COS-7 cells. This suggests that formation of an intrachain disulfide bond is required for SPC-mediated cleavage and that SPC-mediated cleavage is essential to protein function.

Highlights

Lubricin, a protein product of the gene PRG4, is a large (Ͼ200 kDa) secreted proteoglycan that functions as the major lubricant in articulating joints [1, 2]
Neither full-length lubricin nor any smaller polypeptides were present in synovial fluid samples from several patients with camptodactyly-arthropathy-coxa vara-pericarditis syndrome (CACP), equal amounts of synovial fluid protein were evaluated (Fig. 2A)
These antibodies demonstrate that most patients with CACP disease-causing mutations do not have lubricin or lubricin fragments in their synovial fluid (Figs. 2 and 3)

Summary

Introduction

A protein product of the gene PRG4, is a large (Ͼ200 kDa) secreted proteoglycan that functions as the major lubricant in articulating joints [1, 2]. Analysis of the amino acid sequence of lubricin reveals that it is a paralog of vitronectin Both proteins contain somatomedin B (SMB) and hemopexin-like (PEX) domains [6]. Prg knock-out mice exhibit phenotypic features similar to patients with CACP and have severe joint pathology, including articular cartilage destruction and marked synovial cell overgrowth [8]. It appears that lubricin plays an essential role in maintaining healthy joint function and that CACP disease-causing mutations alter the ability of lubricin to perform this role. An allelic series of mutations within PRG4 have been identified in individuals

Methods

Results

Conclusion