Abstract
One of the major secreted proteins of human articular chondrocytes in monolayer or explant culture and of synovial fibroblasts is a glycoprotein with an apparent molecular weight of approximately 39,000, referred to as human cartilage glycoprotein-39 (HC gp-39). The protein was purified, and its complete cDNA sequence was determined. It contained an open reading frame coding for a 383-amino acid long peptide. Comparison of the deduced amino acid sequence with known sequences revealed that HC gp-39 contained regions displaying significant homology with a group of bacterial and fungal chitinases and a similar enzyme found in the nematode, Brugia malayi. In addition significant homologies were observed with three mammalian secretory proteins of as yet unknown function, suggesting that a related protein family exists in mammals. The human protein does not possess any glycosidic activity against chitinase substrates, arguing against any function as an endoglycosidase with specificity for N-acetylglucosamine. Analysis by Northern blotting and by reverse transcription/polymerase chain reaction showed mRNA for HC gp-39 to be present in human articular chondrocytes as well is in liver, while mRNA was undetectable in muscle tissues, lung, pancreas, mononuclear cells, or fibroblasts. Neither the protein nor mRNA for HC gp-39 was detectable in normal newborn or adult human articular cartilage obtained at surgery, while mRNA for HC gp-39 was detectable both in synovial specimens and in cartilage obtained from patients with rheumatoid arthritis. These observations suggest that the expression of HC gp-39 may be related to a response of these cells to an altered tissue environment.
Highlights
One of the major secreted proteinosf human articu- toward maintenanceof this matrix to ensure its proper funclar chondrocytes in monolayer or explant culture and tion
While its biosynthesis in articular cartilage appears to be increased during culture conditions, its expression is not related to thedifferentiation statusof the chondrocytes, since both message as well as thenewly synthesized protein product can be detected in cartilage explants within 24 h of establishment of cultures
4.4 "c 2.4 --C 1.4 "c detectable in RNA preparations of cells freshly isolated from cartilage harvested at autopsy ( i e .after exposure of the tissue to bacterial collagenase for6-8 h). Since both mRNA and the HC gp-39 protein are undetectable in normal cartilage specimens, the induction of HC gp-39 appears to occur rapidly upon changes in the normal tissue environment
Summary
To analyze the regulation of HC gp-39 synthesis, cartilage explants core facility of the Shriners Hospital (Montreal, Quebec) using an and passaged chondrocytes were cultured in the presence of either 50 AB1oligonucleotidesynthesizer (AppliedBiosystems). It program MACAW [26]. Human skin and lung fibroblasts did not to as HC gp-39 is a prominent secretory product of human synthesize and secrete aprotein of equivalent molecular chondrocytes (Fig. 1).In explant cultures of human articular weight, as determined by Western blotting of culture media cartilage, a protein of this size can be detected both in culture from these cells. The sequences used for degenerate oligonucleotide primer design are indicated in bold letters
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