Consequences of disease-causing mutations on lubricin protein synthesis, secretion, and post-translational processing.
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.
Rhee
David K
DK
Department of Genetics and Center for Human Genetics, Case Western Reserve University School of Medicine and University Hospitals of Cleveland, Cleveland, Ohio 44106, USA.
Marcelino
Jose
J
Al-Mayouf
Sulaiman
S
Schelling
Deborah K
DK
Bartels
Cynthia F
CF
Cui
Yajun
Y
Laxer
Ronald
R
Goldbach-Mansky
Raphaela
R
Warman
Matthew L
ML
D020816:Amino Acid Motifs; D000818:Animals; D000906:Antibodies / Q000378:metabolism; D019556:COS Cells; D002522:Chlorocebus aethiops; D004220:Disulfides / Q000737:chemistry; D000939:Epitopes; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007592:Joint Diseases / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D009154:Mutation; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D013577:Syndrome; D013582:Synovial Fluid / Q000737:chemistry; D000099158:Proteoglycan 4
D000906:Antibodies; D004220:Disulfides; D000939:Epitopes; D006023:Glycoproteins; D000099158:Proteoglycan 4