Volume 272, Number 19, Issue of May 9, 1997 pp. 12279-12288
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Structural Analysis of a Tumor-produced Sulfated Glycoprotein Capable of Initiating Muscle Protein Degradation

(Received for publication, October 2, 1996, and in revised form, January 30, 1997)

From the CRC Nutritional Biochemistry Research Group, Pharmaceutical Sciences Institute, Aston University, Birmingham B4 7ET, United Kingdom

A material of M_r 24,000 has been isolated from a cachexia-inducing mouse tumor (MAC16) and shown to initiate protein degradation in isolated gastrocnemius muscle. Biological activity was destroyed by preincubation with peptide N-glycosidase F (PNGase F) and endo--N-acetylgalactosaminidase (O-glycosidase) but not by neuraminidase or trypsin. Antibody reactivity was destroyed by treatment with periodate, indicating carbohydrate moieties to be the antigenic determinants. Antigenic activity was also reduced by treatment with PNGase F and O-glycosidase and was completely destroyed by treatment with chondroitinase ABC but was unaffected by treatment with either trypsin or chymotrypsin, confirming that the N- and O-linked sulfated oligosaccharide chains were both the antigenic and biological determinants.

Biosynthetic labeling of MAC16 cells using a combination of [³⁵S]sulfate and [6-³H]GlcN gave a single component of M_r 24,000 containing both radiolabels. Similar material could not be isolated from a cell line (MAC13) originating from a tumor that does not cause cachexia in vivo. Digestion of ³H/³⁵S material with PNGase F produced two fragments of M_r 14,000 and 10,000 containing both radiolabels, and digestion with O-glycosidase produced three fragments of M_r 14,000, 6,000, and 4,000, the first two contained both radiolabels and the third contained only ³H. Digestion of the fragment of M_r 14,000 released by PNGase F with O-glycosidase also gave fragments of M_r 6,000 and 4,000. The products from both digestions were acidic as determined by anion exchange chromatography on DEAE-cellulose. The negative charge on the fragment of M_r 4,000 was removed by treatment with alkaline phosphatase. This suggests that the charge originated from phosphate residues, and this has been confirmed by biosynthetic labeling of MAC16 cells with [³²P]orthophosphate, where radiolabel was incorporated into material of M_r 24,000 and into the fragment of M_r 4,000 after treatment with O-glycosidase. To determine the size of the polypeptide core MAC16 cells were biosynthetically labeled with L-[2,5-³H]His which after chemical deglycosylation produced a major component of M_r 4,000. These results suggest a model for the M_r 24,000 material consisting of a central polypeptide chain of M_r 4,000 and with phosphate residues that may be attached to the polypeptide or a short oligosaccharide chain containing GlcN, one O-linked sulfated oligosaccharide chain containing GlcN, and of M_r 6,000 and one N-linked sulfated oligosaccharide chain of M_r 10,000 also containing GlcN. Neither chain was cleaved into disaccharides with chondroitinase ABC, suggesting that the material is a sulfated glycoprotein.

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