Expression of membrane‐bound transferrin‐like protein p97 on the cell surface of chondrocytes

Abstract

A concanavalin-A-binding protein of 76 kDa was purified from the plasma membrane fraction of rabbit chondrocyte cultures. Amino acid sequencing of the N-terminal region and of tryptic peptides of the protein, in addition to sequencing of its cDNA revealed that this protein is highly similar to the tumour-associated antigen p97. Hence, it was concluded that this protein is the rabbit form of p97. Western blotting, Northern blotting and reverse-transcription PCR analyses indicated that rabbit p97 is expressed at high levels in cartilage and chondrocytes, but is barely detectable in the bone, liver, kidney, small intestine, eye, pancreas, heart, testis, skeletal muscle, spleen and fibroblasts. Immunocytochemical and immunohistochemical analyses demonstrated that p97 is expressed in the plasma membrane of chondrocytes. p97 transcript was detected in all zones of the cartilage but the level was relatively low in the hypertrophic zone. These findings suggest that p97 is involved in maintaining the cell surface characteristics of chondrocytes.Keywords : chondrocyte ; concanavalin A ; melanotransferrin; membrane-bound transferrin-like protein; p97.Concanavalin A, unlike other plant lectins, stimulates the synthesis of cartilage-matrix proteoglycan (aggrecan) by resting, articular and growth-plate chondrocytes in culture [1]. It also induces the syntheses of alkaline phosphatase and type-X collagen in resting chondrocyte cultures [2]. Concanavalin A induces the synthesis of cartilage in amphibian early gastrula ectoderm [3]. These findings suggest that some concanavalin-A-binding glycoproteins on the cell surface are involved in the control of chondrocyte differentiation.To characterize concanavalin-A-binding membrane proteins in chondrocytes, we labelled surface proteins of chondrocytes with 125 I and tested their concanavalin-A-binding ability. Among them, a 76-kDa protein was a major 125 I-labelled protein and bound to concanavalin-AϪSepharose. Previous studies have shown that a protein of approx. 80 kDa is expressed at very high levels in the outer surface of chick embryo chondrocytes, although it is undetectable in limb mesenchymal cells and dedifferentiated chondrocytes [4]. In the present study, we attempted to purify the 76-kDa concanavalin-A-binding protein and isolateCorrespondence to Y. Kato,

Anti-membrane-bound transferrin-like protein antibodies induce cell-shape change and chondrocyte differentiation in the presence or absence of concanavalin A

“…MTf was purified from rabbit chondrocyte plasma membrane (Kawamoto et al, 1998). Three female BALB/c mice were immunized by a subcutaneous injection of Ribi adjuvant solution (0.2 ml per mouse) containing 20 µg pure MTf.…”

“…The plasma membrane fraction was dissolved in 8 ml of buffer A [10mM Tris/HCl, pH 7.4, 10 µM (p-amidinophenyl) methanesulfonyl fluoride, 10 µM pepstatin A and 1% sodium deoxycholic acid] and applied to a ConA-Sepharose column (1 cm × 3 cm) (Amersham, Piscataway, NJ) equilibrated with buffer A. The ConA-bound glycoprotein was eluted with buffer A containing 0.5 M methyl-α-mannopyranoside as described previously (Kawamoto et al, 1998).…”

“…RNA samples (10 µg) were electrophoresed on a 1% agarose gel containing 2.2 M formaldehyde and transferred to a Hybond-N membrane (Amersham). The membrane was hybridized with a 32 P-labeled rabbit MTf cDNA probe or a 32 P-labeled glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe as described previously (Kawamoto et al, 1998). The membrane was exposed to BioMax X-ray film at -80°C with an intensifying screen.…”

“…Previous studies have shown that MTf is a major ConAbinding protein on the chondrocyte surface (Kawamoto et al, 1998). Because cartilage contains MTf at a much higher level than other normal tissues, chondrogenic cells are a good model for studies on MTf.…”

The melanoma tumor antigen, melanotransferrin (p97): a 25-year hallmark – from iron metabolism to tumorigenesis

“…More recently, there have been reports of MTf protein being identified in normal tissues, including sweat gland ducts (Natali et al, 1987;Alemany et al, 1993), avian eosinophils (McNagny et al, 1996), porcine fetal intestinal epithelial cells (Danielsen and van Deurs, 1995), liver endothelial cells (Sciot et al, 1989;Alemany et al, 1993), rabbit and mouse cartilage (Kawamoto et al, 1998;Nakamasu et al, 1999), avian proximal kidney tubules (McNagny et al, 1996) and the normal endothelium and reactive microglia of brains of Alzheimer's disease (AD) patients Kennard et al, 1996;Rothenberger et al, 1996;Yamada et al, 1999). Normal sera contains very low levels of soluble MTf (sMTf) protein (Brown et al, 1981b) and increased sMTf has been described in patients with AD and arthritis .…”