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

Abstract

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 [35 S]sulfate and [6-3 H]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 3 H/ 35 S 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 3 H. 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 [ 32 P]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-[2,5-3 H]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.

Dermcidin expression in hepatic cells improves survival without N-glycosylation, but requires asparagine residues

“…This contrasts with previous studies, which have suggested heavy N-glycosylation of up to 10 kDa, possibly at one site in the 24 kDa moiety (Todorov et al, 1997), but is consistent with the observation that neither site represents a typical consensus N-glycosylation sequence, although N32 has the characteristics of an atypical site (Welply et al, 1983;Sato et al, 2000). The in vitro translation system was used to assess dermcidin N-glycosylation, as its immunological analysis is problematic.…”

“…This may be essential to its function as a cachetic factor, as dermcidin expression does not appear sufficient to induce cachexia (Monitto et al, 2004). Similarly, deglycosylated PIF lacks proteolytic activity in murine gastrocnemius muscle (Todorov et al, 1996(Todorov et al, , 1997, and in our experiments it was the fully glycosylated PIF molecule that influenced hepatic cell NF-kB and STAT3 activity and gene expression (Watchorn et al, 2001(Watchorn et al, , 2002. However, the PIF aminoacid sequence lacks typical consensus N-and O-glycosylation sites and the mechanism of glycosylation remains unknown.…”

“…The apparent conflict between this evidence and the previous in vitro PIF studies may be accounted for by the use of different antibodies to detect Y-P30, DCD-1 and PIF. The anti-PIF antibody has been shown to recognise a carbohydrate epitope (Todorov et al, 1997) and it is feasible that, when glycosylated, the peptide is not secreted and requires cell lysis for release. We have demonstrated that the dermcidin signal peptide is cleaved and that inhibition of protein transport is required to detect dermcidin in HuH7s, supporting secretion in these cells.…”

“…The specific proteases involved remain to be determined. Thirdly, PIF shares the same core peptide sequence as Y-P30, but is highly glycosylated (Todorov et al, 1997).…”

“…Enzymatic degradation of the PIF glycoprotein has suggested that the molecule consists of a peptide core of molecular weight 4000 Da, which is extensively N-and O-glycosylated to give an apparent mass of 24 kDa (Todorov et al, 1997). This may be essential to its function as a cachetic factor, as dermcidin expression does not appear sufficient to induce cachexia (Monitto et al, 2004).…”