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)

Penio T. Todorov , Melanie Deacon and Michael J. Tisdale Dagger

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

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

A material of Mr 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-alpha -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 Mr 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 Mr 14,000 and 10,000 containing both radiolabels, and digestion with O-glycosidase produced three fragments of Mr 14,000, 6,000, and 4,000, the first two contained both radiolabels and the third contained only 3H. Digestion of the fragment of Mr 14,000 released by PNGase F with O-glycosidase also gave fragments of Mr 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 Mr 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 Mr 24,000 and into the fragment of Mr 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 Mr 4,000. These results suggest a model for the Mr 24,000 material consisting of a central polypeptide chain of Mr 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 Mr 6,000 and one N-linked sulfated oligosaccharide chain of Mr 10,000 also containing GlcN. Neither chain was cleaved into disaccharides with chondroitinase ABC, suggesting that the material is a sulfated glycoprotein.


INTRODUCTION

Depletion of skeletal muscle is an important factor contributing to the decreased survival of cancer patients with loss of cardiac and respiratory muscles being most important. A decreased nutrient intake plays an important role in wasting of lean body mass in cancer cachexia, but it appears that it does not fully account for the changes observed (1). Although protein synthesis is decreased (2), an accelerated protein breakdown accounts in large part for the muscle wasting observed (3).

Several factors have been postulated as signals for this increased muscle proteolysis including tumor necrosis factor-alpha and interleukins 1 and 6 (4, 5). While continuous infusion of the cytokines in vivo has been shown to increase protein degradation in skeletal muscle, none of the cytokines produced a direct stimulation of proteolysis when incubated in vitro (4, 5).

A novel material of Mr 24,000 that appears to fulfill the function of triggering muscle proteolysis during the process of cancer cachexia (6, 7) has been purified from a cachexia-inducing mouse tumor (MAC16). This material was capable of inducing muscle protein degradation in isolated gastrocnemius muscle preparations and of inducing weight loss in vivo and will be referred to as proteolysis inducing factor (PIF).1 Similar, if not identical, material was isolated from the urine of patients with pancreatic carcinoma and weight loss (7).

Structural studies of PIF indicated a short peptide chain of Mr 2,000, which was extensively glycosylated at both Asn and Ser residues (7). Enzymatic degradation suggested that some of the carbohydrate chains contained sulfate residues, whereas lectin blotting (6) indicated the presence of GlcNAc. The material bound strongly to albumin to form a species of Mr 69,000, probably through the sulfate residues which would produce a strong electrostatic linkage. Recent studies (8) show that cell-bound albumin binds peptidoglycan, heparin, and sulfated heparinoids as a complex of Mr 70,000.

The purpose of the present investigation was to characterize PIF in terms of the number, type, and attachment of the carbohydrate chains to the peptide backbone as well as determining the role of the carbohydrate chains in antibody reactivity and protein degradative activity in isolated gastrocnemius muscle.


EXPERIMENTAL PROCEDURES

Materials

RPMI 1640 tissue culture medium with and without L-histidine and fetal bovine serum were from Life Technologies, Inc., Paisley, Scotland, United Kingdom. D-[1,6-3H]GlcN hydrochloride (specific activity 422.3 Ci/mmol) was purchased from DuPont Ltd., Hertfordshire, United Kingdom, and Na235SO4 (specific activity 10-100 mCi/mmol) L-[2,5-3H]histidine (specific activity 56 Ci/mmol), [32P]orthophosphate (10 mCi/ml), and Na125I (1 mCi/10 µl) were purchased from Amersham Int., Bucks, United Kingdom. The Sephadex G-50 and the MW-GF-70 kit used to construct a calibration curve were purchased from Sigma, Poole, Dorset, United Kingdom. The molecular markers were bovine serum albumin, Mr 66,000; carbonic anhydrase, Mr 29,000; cytochrome c, Mr 12,400; and aprotinin, Mr 6,500. PNGase F (recombinant) and O-glycosidase were obtained from Oxford Glycosystems Ltd., Oxfordshire, United Kingdom; neuraminidase and Pronase were from Calbiochem, Nottingham, United Kingdom; keratanase (endo-beta -galactosidase) was from Boehringer Mannheim, East Sussex, United Kingdom; and sulfatase, alkaline phosphate, chondroitinase AC, chondroitinase ABC, pepsin, and peroxidase-conjugated anti-mouse immunoglobulin were from Sigma, Dorset, United Kingdom. Polyvinylchloride assay plates were purchased from Costar, Cambridge, MA and IODO-BEADS were from Pierce. A GlycofreeTM deglycosylation kit was purchased from Oxford Glycosystems, Oxford, United Kingdom. Pure strain female NMRI mice were obtained from our own breeding colony.

Measurement of Protein Degradation

Female NMRI mice were killed by cervical dislocation, and their gastrocnemius muscles were quickly ligated, dissected out, and placed in ice-cold isotonic saline. To minimize diurnal variation and to ensure animals were in the fed state all animals were sacrificed between 9 and 10 a.m. The muscles were blotted, weighed, and carefully tied via tendon ligatures to stainless steel incubation supports (9). This prevents contraction and improves protein balance and energy status (10). Protein degradation was measured by tyrosine release, since tyrosine rapidly equilibrates between intracellular pools and the medium and is neither synthesized nor degraded. Muscles were preincubated in RPMI 1640 (3 ml) lacking phenol red in the presence of serum (280 µl) for 30 min at 37 °C in an atmosphere saturated with O2:CO2 (19:1). The muscles were rinsed and incubated for a further 2 h in Krebs-Henseleit bicarbonate buffer, containing 6 mM D-glucose, 1.2 mg/ml bovine serum albumin, and 130 µg/ml cycloheximide with continuous gassing. At the end of the incubation the buffer was removed, deproteinized with ice-cold 30% trichloroacetic acid (0.2 ml), centrifuged at 2800 × g for 10 min, and the supernatant used for the measurement of tyrosine by a fluorimetric method (11) at 570 nm on a Perkin-Elmer LS-5 luminescence spectrometer.

Cell Culture and Radiolabeling Procedures

The two cell lines MAC16 and MAC13 were derived from the solid tumors grown in vivo and were a gift from Prof. J. A. Double and Dr. M. Bibby (University of Bradford, Yorkshire, United Kingdom). The MAC16 cells were microbiologically free and grew in suspension, while the MAC13 grew as a monolayer in RPMl 1640 medium containing 5% fetal bovine serum under an atmosphere of 5% CO2 in air. Cells were grown in progressively lower fetal bovine serum concentrations, and a level of 1.5% was used for the labeling experiment. Cells (108) were resuspended in RPMI 1640 (100 ml) containing 1.5% dialyzed fetal calf serum and Na235SO4 (1 µCi/ml), and [3H]GlcN (2 µCi/ml) or [3H]His (5 µCi/ml) or [32P]orthophosphate (2 µCi/ml) was added. The cells were incubated under an atmosphere of 5% CO2 in air at 37 °C for 48 h and sedimented by low speed centrifugation (1500 rpm for 5 min on a bench-top centrifuge). The cell pellet was resuspended in 1 ml of 10 mM Tris·HCl, pH 8.0, containing 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 0.5 mM EGTA, and 1 mM dithiothreitol and dissociated using an ultrasonic oscillator. Debris was removed by centrifugation (15,000 rpm for 20 min), and solid ammonium sulfate (80% w/v) was added to the supernatant, and the mixture was stored overnight at 4 °C. The precipitated proteins were collected by centrifugation, and the pellet was resuspended in 10 mM Tris·HCl, pH 8.0, containing 0.5 mM PMSF, 0.5 mM EGTA, and 1 mM dithiothreitol, and salt was removed by ultrafiltration with an Amicon filtration cell containing a membrane filter with a molecular weight cut-off of 10,000 against the same solution. The concentrated sample was loaded onto an affinity column containing mouse monoclonal antibody (6) coupled to Affi-Gel Hz (Bio-Rad, Hemel Hempstead, United Kingdom) equilibrated with 10 mM Tris·HCl, pH 8.0. After overnight circulation at a flow rate of 5 ml/h, the column was washed with 10 mM Tris·HCl, pH 8.0, and the retained material was eluted with 100 mM glycine HCl, pH 2.5, into tubes containing 1 M Tris·HCl, pH 8.0, for neutralization. The fractions were counted for radioactivity using a Packard 2000 CA liquid scintillation analyzer.

Enzymatic and Chemical Deglycosylation

Enzyme treatments were performed as follows: PNGase F (1 unit/20 µl) for 24 h at 37 °C in 20 mM phosphate, pH 7.5, containing 50 mM EDTA and 0.2 mM PMSF; O-glycosidase (1 milliunit/20 µl) for 20 h at 37 °C in 100 mM phosphate/citrate, pH 6.0; neuraminidase (1.2 milliunits/ml) for 16 h at 37 °C in 50 mM sodium acetate, pH 5.0, containing 2 mM CaCl2; sulfatase (type H-1 from Helix pomatia), (16 units/ml) for 24 h at 37 °C in 200 mM sodium acetate, pH 5.0; endo-beta -galactosidase for 20 min at 37 °C in 50 mM sodium acetate, pH 5.8; alkaline phosphatase (5 units) for 18 h at 37 °C in 0.5 M NH4HCO3, pH 8.5; chondroitinase AC (1 unit/ml) for 18 h at 37 °C in 50 mM Tris·HCl, pH 8.0, containing 50 mM NaCl; chondroitinase ABC (2 units/ml) for 18 h at 37 °C in 0.25 M Tris·HCl, pH 8.0. In all cases buffers contained 0.5 mM PMSF and 1 mM dithiothreitol to inhibit proteolytic degradation. Trypsin (300 µg/ml), chymotrypsin (300 µg/ml), and pepsin (200 µg/ml) were incubated for 1 h at 37 °C in 10 mM Tris·HCl, pH 8.0, containing 2 mM CaCl2 and Pronase (predigested for 10 min at 37 °C) for 1 h at 37 °C (4.3 units/ml) in 100 mM Tris·HCl, pH 8.0. Both chondroitinase AC and ABC were assayed before use using chondroitin A as substrate. The reaction rate was monitored by measuring the increase in absorbance at 232 nm. Enzyme activity calculated from the initial rate and using E = 3800 M-1 for reaction products at pH 8 was 2.85 and 2.70 µmol/min for 1 unit of chondroitinase ABC and AC, respectively. Chemical deglycosylation by anhydrous trifluoromethane sulfonic acid was achieved with a GlycofreeTM deglycosylation kit according to the manufacturer's instructions. Nitrous acid deamination was carried out on the sample (in 50 µl of water) kept at room temperature for 10 min. The products of the reaction were then analyzed on a column of Sephadex G-50.

Enzyme-linked Immunosorbent Plate Assay

Samples were divided into two and immobilized on a 96-well polyvinylchloride assay plate overnight at 4 °C. The liquid was removed by aspiration, and the wells were washed three times with PBS + 0.1% Tween 20 (200 µl). Blocking solution (200 µl of PBS containing 0.1% Tween 20 and 3% bovine serum albumin) was added to the wells, and the plate was incubated for 2 h at 37 °C. One-half of the sample was incubated with the monoclonal antibody (10 µg/ml) in blocking solution (100 µl) for 1 h at room temperature, while the other half was incubated in the same solution but in the absence of the antibody. After removal of the antibody solution the wells were washed six times before the addition of a peroxidase-conjugated anti-mouse immunoglobulin, diluted 1 in 500 in blocking solution (100 µl/well), and the plates were incubated for a further 1 h at 37 °C. The wells were washed six times, and then the substrate solution, o-phenylenediamine dihydrochloride (0.04%), hydrogen peroxide (0.012%) in 0.15 M phosphate citrate buffer, pH 5.0 (100 µl/well), was added for 30 min. The reaction was terminated by the addition of 0.2 M H2SO4 (50 µl/well), and the absorbance was determined at 492 nm using a microplate reader (Anthos Labtec Instruments).


RESULTS

When isolated mouse gastrocnemius muscle was incubated with serum from mice bearing the MAC16 tumor and with a weight loss between 2 and 4.4 g an increased protein degradation was observed as measured by tyrosine release (Table I). This effect was attenuated by incubation of the serum with monoclonal antibody prior to addition to the muscle preparation. An increased tyrosine release could also be produced by addition of affinity purified antigen to serum from non-tumor-bearing mice (Table I), thus confirming that this material was the serum component responsible for the degradation of skeletal muscle proteins. The increased tyrosine release produced by the affinity purified antigen was abolished by preincubation with PNGase F, O-glycosidase, and sulfatase but unaffected by treatment with neuraminidase or trypsin. These results suggest that protein degradation is mediated by N- and O-linked carbohydrate chains in the molecule.

Table I. Effect of affinity purified antigen (Ag) and serum from MAC16 mice with established cachexia on tyrosine release from gastrocnemius muscle

Solid MAC16 tumors excised from mice with established cachexia were fractionated on an affinity column containing the MAC16 monoclonal antibody as described (6). The enzyme-linked immunosorbent assay-positive fractions were pooled and concentrated, and portions (013A492 units 0.77 µg of protein) were treated with the enzymes as described under "Experimental Procedures." All samples were preincubated with serum from non-tumor-bearing mice for 30 min at 37 °C prior to determination of tyrosine release from gastrocnemius muscle as described under "Experimental Procedures." After incubation with trypsin (0.2 µg/µg of protein) for 20 h; alpha 1-antitrypsin (8.3 µg) was added prior to addition to serum. Using this procedure tyrosine release when trypsin was added to serum did not differ significantly from that observed with normal mouse serum. Solid MAC16 tumors excised from mice with established cachexia were fractionated on an affinity column containing the MAC16 monoclonal antibody as described (6). The enzyme-linked immunosorbent assay-positive fractions were pooled and concentrated, and portions (013A492 units 0.77 µg of protein) were treated with the enzymes as described under "Experimental Procedures." All samples were preincubated with serum from non-tumor-bearing mice for 30 min at 37 °C prior to determination of tyrosine release from gastrocnemius muscle as described under "Experimental Procedures." After incubation with trypsin (0.2 µg/µg of protein) for 20 h; alpha 1-antitrypsin (8.3 µg) was added prior to addition to serum. Using this procedure tyrosine release when trypsin was added to serum did not differ significantly from that observed with normal mouse serum.
Treatment Tyrosine

nmol/mg/2 h
Normal mouse serum 37  ± 2
Cachectic mouse serum 54  ± 6a
Cachectic mouse serum + antibody 35  ± 3
Normal mouse serum + Ag 67  ± 18b
Normal mouse serum + Ag + PNGase F 28  ± 3c
Normal mouse serum + Ag + sulfatase 41  ± 4d
Normal mouse serum + Ag + O-glycosidase 35  ± 4c
Normal mouse serum + Ag + neuraminidase 65  ± 3
Normal mouse serum + Ag + trypsin 62  ± 10

a p < 0.05 when compared with normal mouse serum.
b p < 0.02 when compared with normal mouse serum.
c p < 0.02 when compared with normal mouse serum + Ag.
d p < 0.05 when compared with normal mouse serum + Ag.

A similar relationship was obtained for antigen binding activity (Table II). Immunological activity was completely destroyed by treatment with periodate, indicating that the carbohydrate moieties are involved in the epitope. Antibody binding activity was inhibited by PNGase F, O-glycosidase, and sulfatase (Table II) but unaffected by treatment with neuraminidase or chymotrypsin, indicating that N- and O-linked carbohydrate chains are the antigenic determinants. In addition antigen reactivity was reduced by treatment with chondroitinase AC and completely destroyed by treatment with chondroitinase ABC but was unaffected by treatment with endo-beta -galactosidase. Since the latter enzyme only hydrolyzes internal beta -galactoside linkages of oligosaccharides having non-sulfated beta -galactose residues, these results suggest that the principal immunological determinants reside in sulfated oligosaccharide chains and that the material is a glycoprotein or proteoglycan.

Table II. Studies of antigenic determinants of proteolysis-inducing factor


Treatment Antigen binding activitya (A492)

None 0.24  ± 0.031
Trypsin 0.17  ± 0.021
Chymotrypsin 0.21  ± 0.006
Neuraminidase 0.29  ± 0.017
O-Glycanase 0.11  ± 0.016
PNGase F 0.16  ± 0.014
Keratanase 0.26  ± 0.003
Sulfatase 0.11  ± 0.02
Chondroitinase AC 0.17  ± 0.02
Chondroitinase ABC 0.01  ± 0.01
Alkaline phosphatase 0.13  ± 0.01

a Values given are mean ± S.E. for three determinations per point.

To obtain information on the nature of the linkage and the number and types of glycan chains, MAC16 cells were doubly labeled with [35S]sulfate and [3H]GlcN, and the antigen was purified by ammonium sulfate fractionation and affinity chromatography. The elution profile from the affinity column showed a single band of radioactivity containing both radiolabels (Fig. 1A), which represented 13% of the 35S and 15% of the 3H radiolabel from the ammonium sulfate precipitate. This material showed a single band of radioactivity corresponding to a Mr of 24,000 on SDS-PAGE (Fig. 1B). Similar material could not be isolated from a cell line (MAC13) originating from a tumor that does not produce cachexia in vivo. Fractionation of the MAC16 material on a Sephadex G-50 column under dissociating conditions confirmed a single band of Mr 24,000 that contained both 35S and 3H (Fig. 2A). The Mr of this material was the same as that previously isolated from the MAC16 tumor using a combination of affinity and reverse phase hydrophobic chromatography (7). Identical sham incubations (without PNGase F) showed no radioactivity eluting at positions corresponding to the released material. Re-chromatography of this material after overnight digestion with PNGase F gave two bands of radioactivity eluting at positions corresponding to Mr of 14,000 and 10,000 (Fig. 2B). Both fragments contained 3H and 35S. Digestion of the Mr 24,000 material with O-glycosidase and fractionation on Sephadex G-50 showed conversion to three bands of radioactivity corresponding to Mr of 14,000, 6,000, and 4,000 (Fig. 2C). Incubation with buffer in the absence of O-glycosidase showed no degradation of the Mr 24,000 material. The first two bands contained both 3H and 35S, and the band of Mr 4,000 contained only 3H. Treatment of the material of Mr 14,000 produced from PNGase F digestion with O-glycosidase converted it into two fractions corresponding to Mr of 6,000 and 4,000 (Fig. 2D).


Fig. 1. A, elution profile of radioactivity bound to an affinity column after biosynthetic labeling of MAC16 cells with [35S]sulfate (bullet ) and [6-3H]GlcN (open circle ). The details of the procedure are given under "Experimental Procedures." B, autoradiograph of material shown in A after SDS-PAGE electrophoresis. Lane 1, from tissue culture supernatant; lane 2, from MAC16 cells.
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Fig. 2. Elution profile of affinity purified material shown in Fig. 2 on a Sephadex G-50 column (40 × 1.5 cm) equilibrated with 10 mM Tris·HCl, pH 7.0, containing 0.2 M NaCl and eluted at 6 ml/h. Fractions (0.7 ml) were collected and assayed for 35S (bullet ) and 3H (open circle ) radioactivity using a dual counting procedure. A, without treatment. B, after 24 h treatment with recombinant PNGase F at pH 8.0. C, after 20 h incubation with O-glycosidase at pH 6.0. D, enzymatic digestion of the fragment of Mr 14,000 shown in B with O-glycosidase. Treatment with the incubation buffers alone had not effect on the elution position of the Mr 24,000 material on the Sephadex G-50 column. The PNGase F was free of protease, endo-alpha -N-acetylgalactosaminidase H/endo-alpha -N-acetylgalactosaminidase F, and did not contain glycosidase activity. The O-glycosidase was also free of protease and contaminating glycosidases.
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To determine the acidity of the charged groups on each of the oligosaccharide chains, the Mr 24,000 material was subjected to enzymatic digestion as above, and the products were fractionated on a DEAE-cellulose column under the influence of a linear gradient from 0 to 0.3 M NaCl. The identity of the eluted peaks was determined by exclusion chromatography on Sephadex G-50. Anion exchange chromatography after digestion with PNGase F gave two bands eluting at 0.20 M NaCl (Mr 10,000) and 0.28 M NaCl (Mr 14,000) (Fig. 3). Digestion with O-glycosidase gave three bands that all adhered to the DEAE-cellulose column in order of elution 0.20 M NaCl (Mr 6,000), 0.24 M NaCl (Mr 4,000), and 0.29 M NaCl (Mr 14,000) (Fig. 4). In both cases there was no further fractionation between the 3H- and 35S-labeled oligosaccharide chains. Although the O-linked oligosaccharide chain of Mr 4,000 did not contain sulfate residues as determined by biosynthetic labeling, it was more acidic than the sulfated chains of Mr 6,000 and 10,000, as determined by the concentration of NaCl required to elute it from the DEAE-cellulose column.


Fig. 3. Elution profile of radioactivity determined as 35S (bullet ) and 3H (open circle ) in the oligosaccharide chains released after enzymatic deglycosylation with PNGase F (A) and fractionation by high performance liquid chromatography on a DEAE-cellulose column (Applied Biosystems Inc.) under the influence of a NaCl gradient from 0 to 0.3 M NaCl. Affinity purified biosynthetically labeled material (Fig. 2) was subjected to enzymatic deglycosylation as described under "Experimental Procedures," and the products were desalted using a microcon microconcentrator containing a filter with a molecular size cut-off of 10,000 (Amicon Corp.) and fractionated on a DEAE-cellulose column. The flow rate was 0.2 ml/min with solvent system A, 10 mM sodium phosphate buffer, pH 5.3, and solvent system B, 10 mM sodium phosphate containing 0.3 M NaCl. The gradient was for 10 min at 0% B, 40 min at 100% B, 50 min at 100% B, and 60 min at 0%t B. Absorbance was monitored at 214 nm, and the radioactivity of the individual fractions was determined using a dual counting procedure. The bands eluting at 0.28 M NaCl (B) and 0.20 M NaCl (C) were further fractionated on a Sephadex G-50 column.
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Fig. 4. Elution profile of radioactivity determined as 35S (bullet ) and 3H (open circle ) in the oligosaccharide chains released after enzymatic deglycosylation with O-glycosidase (A). The bands eluting at 0.29 M NaCl (B), 0.20 M NaCl (C), and 0.24 M NaCl (D) were further fractionated on a Sephadex G-50 column. The conditions for the DEAE-cellulose column are given in the legend to Fig. 3.
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The nature of the charged groups on this non-sulfated oligosaccharide chain was investigated by digestion with either neuraminidase or phosphatase followed by fractionation of the products by anion exchange chromatography. After treatment of the oligosaccharide with neuraminidase, the product still adhered to the DEAE-cellulose column and eluted at 0.24 M NaCl, suggesting the absence of sialic acid residues. However, after incubation with alkaline phosphatase the acid group was removed and the product no longer attached to the anion exchange column, although the apparent Mr was not affected (results not shown). The Mr of the intact molecule was also unaffected by alkaline phosphatase as determined by exclusion chromatography on Sephadex G-50. This suggests that phosphate residues are responsible for the negative charge on the O-glycosidase digestion product of Mr 4,000. Antibody binding activity of the Mr 24,000 material was reduced by 53% after treatment with alkaline phosphatase (Table II), and immunoreactivity on Western blotting was completely destroyed, suggesting that the phosphate residues are also important antigenic determinants. To confirm the presence of phosphate groups on the chain of Mr 4,000, MAC16 cells were biosynthetically labeled with [32P]orthophosphate, and the antigen was purified by ammonium sulfate precipitation and affinity chromatography as before. The 32P was confined to a single band of Mr 24,000 as determined by SDS-PAGE (Fig. 5A) and exclusion chromatography on Sephadex G-50 (Fig. 5B). Treatment with PNGase F (Fig. 5, A and C) or chondroitinase AC (Fig. 5A) yielded a single fraction containing 32P of Mr 14,000. Digestion of the material of Mr 24,000 with either O-glycosidase (Fig. 5, A and D) or chondroitinase ABC gave a single band containing 32P of Mr 4,000. This material adhered to a DEAE column and was eluted with 0.24 M NaCl (Fig. 5E) as did the fragment from material labeled with [3H]GlcN (Fig. 3).


Fig. 5.

A, autoradiograph of material, obtained by biosynthetic labeling of MAC16 cells with [32P]orthophosphate and purification by affinity chromatography, on a 16.5% Tricine/SDS-PAGE gel. Lane 1, without treatment; lane 2, with chondroitinase AC; lane 3, with PNGase F; lane 4, with O-glycosidase; lane 5, with chondroitinase ABC. B, elution profile of material from MAC16 cells on a Sephadex G-50 column after biosynthetically labeling with either 32P (bullet ) or [3H]His (open circle ) and purified by affinity chromatography; C, after treatment with PNGase F; D, with O-glycosidase. E, elution profile of radioactivity, determined either as 32P (bullet ) or 3H (open circle ) on a DEAE-cellulose column, as described in the legend to Fig. 4, of the fragment released by treatment with O-glycosidase as shown in D.


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To determine the size of the polypeptide core, MAC16 cells were labeled with L-[2,5-3H]His, which is the amino acid at residue 16 of the core (6, 7). The radiolabel was incorporated into a single component of Mr 24,000 after fractionation of the cell extract by affinity chromatography (Fig. 5B; Fig. 6). Treatment with PNGase F showed 3H labeling of the fragment of Mr 14,000 (Fig. 5C; Fig. 6, the lower band in the autoradiograph may be an artifact). Treatment with O-glycosidase showed 3H labeling of the Mr 4,000 fragment (Fig. 5D; Fig. 6). This fragment was the same as that found after biosynthetic labeling with 32P since both adhered to a DEAE-cellulose column and were eluted with 0.24 M NaCl (Fig. 5E). Chemical deglycosylation with anhydrous trifluoromethanesulfonic acid showed a major band with a Mr near 4,000 but lower than that formed by treatment with O-glycosidase (Fig. 6) and a minor band at Mr 2,000. This suggests that the molecular weight of the polypeptide core is 4,000 and that there may be a short oligosaccharide chain (labeled with [3H]GlcN) which is phosphorylated or the peptide chain itself may be phosphorylated.


Fig. 6. Autoradiograph of material, obtained by biosynthetic labeling of MAC16 cells with [3H]His and subsequent purification by affinity chromatography, on a 16.5% Tricine/SDS-PAGE gel. Lane 1, without treatment; lane 2, with PNGase F; lane 3, chemical deglycosylation with a Glycofree deglycosylation kit; lane 4, with O-glycosidase.
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To determine the nature of the oligosaccharide chains, material of Mr 10,000, released by treatment of [35S]sulfate- and [3H]GlcN-labeled Mr 24,000 with PNGase F, was treated with chondroitinase ABC followed by re-chromatography on a Sephadex G-50 column. Two bands of radioactivity corresponding to Mr of 8,000 and 2,000 were obtained (Fig. 7A) but no low molecular weight material corresponding to disaccharides. Treatment of the oligosaccharide chain of Mr 6,000 produced by cleavage with O-glycosidase with either chondroitinase AC or ABC (Fig. 7B) had no effect on the molecular weight. These results suggest that material of Mr 24,000 does not contain glycosaminoglycan chains and that it is a sulfated glycoprotein rather than a proteoglycan. This conclusion was substantiated by analysis of the carbohydrate chains released after treatment with 0.1 N NaOH and 2 M NaBH4 for 16 h. Material of Mr 24,000 doubly labeled with [35S]sulfate and [3H]His released fragments of Mr 14,000, 6,000, and 4,000 (Fig. 7D). The latter fragment was labeled only with 3H, suggesting that it represented the polypeptide core. The elution positions of both the fragments of Mr 14,000 and 6,000 were not affected by further treatment with chondroitinase ABC, AC, or nitrous acid (data not shown) again confirming that the oligosaccharide chains were not of the chondroitin, dermatan, or heparan sulfate type.


Fig. 7.

Elution profile of radioactivity determined as 35S (bullet ) and 3H (open circle ) on Sephadex G-50 of the oligosaccharide chains released after enzymatic deglycosylation with PNGase F (A) and O-glycosidase (B) after incubation with chondroitinase ABC (0.2 units in 0.1 ml) for 18 h at 37 °C in 250 mM Tris·HCl, pH 8.0. C, elution profile of radioactivity on Sephadex G-50 from affinity purified material of Mr 24,000 biosynthetically labeled with 35SO4 (bullet ) and [3H]His (open circle ) after incubation with 2 M NaBH4 in 0.1 M NaOH for 16 h at 37 °C. Excess NaBH4 was destroyed with 0.25 M acetic acid in methanol, and the boric acid was removed by evaporation of the methanolic solution under a stream of nitrogen. The procedure was repeated twice with the same amount of acidified methanol and twice with methanol alone.


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DISCUSSION

The data in the present study provide strong support that protein degradation in skeletal muscle during the process of cancer cachexia is mediated by a tumor-produced sulfated glycoprotein of Mr 24,000. This material could be responsible for the accelerated breakdown of isolated rat diaphragm muscle observed when incubated with plasma from cancer patients with weight loss greater than 10% (12), since we have shown similar material in both human colonic carcinomas and in the urine of patients with pancreatic carcinoma and established cachexia (7). Although interleukin-Ialpha and interleukin-Ibeta were shown to stimulate protein catabolism in the rat diaphragm muscle bioassay, in the study of Belizario et al. (12) antibodies to the recombinant human cytokines gave only partial neutralization of bioactivity in less than half of the patients investigated, suggesting other active factors that could not be defined. The structure of the Mr 24,000 material is novel, and the peptide sequence (6, 7) is distinct from the recognized cytokines. Antisera to this material was not cross-reactive with the cytokines' tumor necrosis factor-alpha and interleukin-6. In addition the ability of MAC16 cells to produce this material in vitro, as demonstrated by biosynthetic labeling studies, confirms that it arises from tumor rather than host cells. A histologically related cell line, MAC13, derived from a non-cachexia-inducing tumor was found not to be capable of producing radioactively labeled Mr 24,000 material using either [6-3H]GlcN or Na235SO4 as the precursors. This suggests that it is only produced by some tumor cell lines which are capable of inducing cachexia.

Both functional and immunological studies provide evidence that biological activity is mediated through N- and O-linked oligosaccharide chains in the molecule. To identify the size and position of attachment of the oligosaccharide chains as well as the size of the polypeptide core, MAC16 cells have been biosynthetically labeled with 35SO4, [3H]GlcN, [3H]His, and 32Pi followed by affinity purification to isolate the labeled product. In all cases a single component of Mr 24,000 was obtained as determined by SDS-PAGE and exclusion chromatography. Similar results have previously been obtained with high performance liquid chromatography-purified material labeled with Na125I (7).

With regard to the molecular weight of the material and the fragments generated by enzymatic deglycosylation, these are apparent rather than real, since glycosylated molecules have hydrodynamic volumes that differ per unit of molecular weight from those of globular proteins, which have been used as molecular weight standards.

With this caveat incubation with recombinant protease-free PNGase F, which specifically cleaves the GlcNAc-Asn bond of N-linked oligosaccharides (13), yielded two fragments one of Mr 14,000 and one of Mr 10,000. The former fragment contained the peptide chain since it contained [3H]His (Fig. 6) or 125I, when Na125I was used (7). The latter fragment contained only 35S and 3H when 35SO4 and [3H]GlcN were used in biosynthetic labeling studies (Fig. 2B), suggesting that it is an oligosaccharide chain. Retention of 35S and 3H in the Mr 14,000 fragment suggests that it also contains an oligosaccharide chain. Chondroitinase AC also cleaved material of Mr 24,000 into the fragment of Mr 14,000 (Fig. 5) and caused the same decrease in antibody binding activity as PNGase F. However, the fragment of Mr 14,000 was not cleaved into lower molecular weight material by chondroitinase AC or ABC and did not release any of the 35SO4 label with nitrous acid, whereas the oligosaccharide chain of Mr 10,000 formed two products with chondroitinase ABC of Mr 8,000 and 2,000 (Fig. 7A). These results suggest the absence of N-sulfate residues and the lack of formation of disaccharides with chondroitinase AC or ABC suggest that the oligosaccharide chains were not of the chondroitin or dermatan sulfate type. The presence of sulfate in N-linked oligosaccharides has been reported in several proteins including the low density lipoprotein receptor (14), ovalbumin (15), and pituitary hormones (16, 17).

Treatment of material of Mr 24,000 with O-glycosidase yielded two fragments of Mr 6,000 and 4,000. Material biosynthetically labeled with 35SO4 and [3H]GlcN showed the two labels to be incorporated into the fragment of Mr 6,000, suggesting that it was an O-linked glycan chain. O-Glycosidase has stringent specificity for the core structure Galbeta 1right-arrow3 GalNAc alpha 1-Ser/Thr and will not cleave any other O-linked glycan. Thus the sulfoglycan of Mr 6,000 could not be a typical chondroitin sulfate chain with xylose O-glycosidically linked to serine (18). This conclusion is substantiated by lack of cleavage of the oligsaccharide fragment by either chondroitinase ABC or AC. Thus the material of Mr 24,000 does not appear to contain glycosaminoglycan chains attached to either Asn or Ser residues and is therefore a sulfated glycoprotein rather than a proteoglycan.

Treatment of the fragment of Mr 14,000, obtained by digestion of material of Mr 24,000 with PNGase F, with O-glycosidase also yielded the two fragments of Mr 6,000 and 4,000. Although only material of Mr 6,000 was labeled with 35S, both fragments were acidic as determined by the binding to a DEAE-cellulose column and subsequent elution with NaCl. The charge on the fragment of Mr 4,000 was removed by treatment with alkaline phosphatase, suggesting that the acidic group was phosphate. This was confirmed by biosynthetically labeling MAC16 cells with [32P]orthophosphate, which led to 32P incorporation into the glycoprotein of Mr 24,000, the PNGase F cleavage fragment of Mr 14,000, and the O-glycosidase cleavage fragment of Mr 4,000 (Fig. 6). Biosynthetic labeling of the polypeptide core with [3H]His also showed incorporation of the radiolabel into the same fragments (Fig. 6). This suggests that the phosphate residues are attached to the peptide core or a short oligosaccharide chain containing GlcN attached to the peptide core.

Amino acid sequence studies showed a short polypeptide core containing 18 (20) amino acids (6, 7). Attempts to determine the molecular weight of the polypeptide have yielded conflicting results. Thus chemical deglycosylation of the iodinated material using anhydrous trifluoromethanesulfonic acid gave a single band of Mr 2,500 (7). However, chemical deglycosylation using material that had been biosynthetically labeled with [3H]His showed a strong band at Mr 4,000 and a weaker one at Mr 2,000. The latter result suggests that the polypeptide chain may be longer that that previously reported.

These results suggest a model for the Mr 24,000 glycoprotein consisting of a central polypeptide chain and a short oligosaccharide chain containing GlcN and with phosphate residues and of Mr 4,000, one O-linked sulfated oligosaccharide chain containing GlcN and of Mr 6,000, and one N-linked sulfated oligosaccharide chain of Mr 10,000 also containing GlcN. The apparent difference between the sum of the molecular weight of the subunits and that of the whole molecule may be due to differences in the true molecular weight of the carbohydrate fragments with that achieved using globular proteins as molecular weight standards. The high negative charge on all the fragments may also lead to disparities in calculating the exact molecular weight.

The sequence contained a single Asn residue, which must be the site for N-glycosylation (7). The sequence NXS is an N-glycosylation sequon, although the presence of proline in the middle of the sequon has been suggested to inhibit the attachment of sugars (19). Thus it is possible that the Asn that is glycosylated is downstream of that in the sequence reported. There are three potential O-glycosylation sites at Ser residues 7, 11, and 15. (7). Low levels of Ser were recovered at all positions, and residue 15 often yielded a blank, strongly suggesting O-glycosylation at this site, since hydrophobic amino acid derivatives are not efficiently extracted in the non-polar solvent used after cleavage in the Edman degradation (20).

The biological significance of sulfate esters on O- and N-linked oligosaccharide chains is presently unknown. In thyroglobulin sulfated complex N-linked oligosaccharides may serve as recognition signals in directing the intracellular traffic, follicular secretion, or reabsorption (21). Sulfate residues would also provide a strong electrostatic linkage to cellular receptors leading to tight binding. It is not known why the Mr 24,00 glycoprotein should be expressed only in tumor cells capable of producing cachexia. There are quantitative and qualitative changes in the expression of acidic glycoconjugates in colon cancer, which are associated with progression and metastasis (22, 23). In addition to differential expression of the core polypeptide in tumors producing cachexia, there may be differences in expression of glycosyl and sulfotransferases. Considering the complexity of structure of this material it is not surprising that only certain tumor cells have the enzymatic machinery capable of production. However, the conservation of structure between mouse and man (7) suggests that this material may be important for tumor function.


FOOTNOTES

*   This work was supported by Cancer Research Campaign Grant SP1518.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom requests for reprints should be addressed.
1   The abbreviations used are: PIF, proteolysis inducing factor; PNGase F, peptide: N-glycosidase F; O-glycosidase, endo-alpha -N-acetylgalactosaminidase; PMSF, phenylmethylsulfonyl fluoride; PBS, Ca2+- and Mg2+-free phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl) ethyl]glycine.

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