Metastatic Melanoma Cell Neparanase

Vol. 259, No. 4, Issue of February 25, pp. 2283-2290 1984 Printed in d.S.A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY 01984 by The American Society of Biological Cbemista, Inc.

Metastatic MelanomaCell Neparanase CHARACTERIZATION OF HEPARAN SULFATE DEGRADATION FRAGMENTSPRODUCED BY B16 MELANOMA ENDOGLUCURONIDASE* (Received for publication, August 30, 1983)

Motowo Nakajimazg, Tatsuro Irimural7, Nicola Di FerrantelJ**, and GarthL. Nicolson$Sf: From the $Department of Tumor Biology, The University of Texas-M.D. Anderson Hospital and Tumor Institute, Houston, Texas 77030 and the )/Laboratoriesof Connective Tksue Research, Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030

.

easily penetrate intactblood vessel walls (3,4) and endothelia1 basal lamina-like matrix(5-7) composed of glycoproteins such as fibronectin (8-10) and laminin (11-13), collagens, particularly type IV (14-16), and sulfated proteoglycans (17-19). It is thought that metastatic cells penetrate theendothelial basal lamina using degradative enzymes specific for these basal lamina components (1, 2, 20-23). Vascular endothelial cell extracellular matrix has been used as a substrate for tumor cell degradation studies (6, 17-19, 24). Highly metastatic cells solubilize the major components of endothelia1 basal lamina such as fibronectin, laminin, and HS’ (6, 17-19,24). We have found that theaverage M,of the HS released from endothelial basal lamina-like matrixby B16 melanoma cells is approximately one-third the M, of the original molecules in the untreated matrix, suggesting that B16 cells possess an endoglycosidase capable of degrading HS into intermediate M, fragments (17). Highly invasive and metastatic B16 sublines degrade sulfated glycosaminoglycans (S-GAGS)of the basal lamina-like matrix at higher rates than B16 cells of lower metastatic potential (6, 18). B16 melanoma cells also fragment purified HS (18, 19). Intact B16 cells or B16 cell extracts from sublines of high lung colonization potential degrade purified HS at higher rates than B16 cells of poor lung colonization potential (18). Here we demonstrate that these activities are due to a HSspecific endoglucuronidase in €316 melanoma cells by characterization of HS degradation products using gel chromatography (25-27) and high speed gel permeation chromatography ( 2 8 ) and by determining the reducing terminal saccharides of IiS degradation fragments. EXPERIMENTAL PROCEDURES

During blood-borne tumor metastasis formation, malignant cells must invade the vascular endothelial cell layer and its underlying basal lamina ( 1 , 2 ) .In vitro metastatic tumor cells

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 5 Supported by American Cancer Society Institutional Grant IN34. II Supported by American Cancer Society Institutional Grant IN121B. ** Supported by Research Grant R01-AM26482 from the National Institutes of Health, United States Public Health Service. $3 Supported by Research Grants R01-CA28844 and R01CA28867 from the National Institutes of Health, United States Public Health Service. To whom correspondence should be addressed at Department of Tumor Biology-108, The University of Texas-M.D. Anderson Hospital and Tumor Institute,6723 Bertner Avenue, Houston. Texas 77030.

Materials CeMs and Cell Culture-Highly invasive and lung metastatic murine B16 melanoma subline (B16-BL6)was obtained from Dr. I. J. Fidler (The University of Texas-M.D. Anderson Hospital and Tumor Institute, Houston, TX). Melanoma cells weregrownon plastic tissue culture dishes in a 1:l mixture of DME/FlP (Gibco, Grand Island, NY), supplemented with 5% heat-inactivated fetal bovine serum (Reheis, Kankakee, IL),under humidified conditions with 95% air5%CO,. BAE cells, obtained from Dr. D. Gospodarowicz (University of California, Medical Center, San Francisco, CA), were cultured in

The abbreviations used are: HS, heparan sulfate; GAG($, glycosaminoglycan(s); C4S, chondroitin 4-sulfate; C6S, chondroitin 6-SUIfate; DS, dermatan sulfate; HA, hyaluronic acid; KS, keratan sulfate; SAL, D-saccharic acid l,4-lactone; BAE cells, bovine aortic endothelial cells; BCE cells, bovine corneal endothelial cells; DME, Dulbecco’s modified minimum essential medium; F12, Ham’s F-12 medium DPBS, Dulbecco’s phosphate-buffered saline; EHS, EngelbrethHolm-Swarm; PYS, parietal yolk sac.

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Heparan sulfate(HS), a prominent component of vascular endothelial basal lamina, is cleaved into largeM, fragments and solubilized from subendothelial basal lamina-like matrix by metastatic murine B16 melanoma cells. We have examined the degradation products of HS and otherpurified glycosaminoglycansproduced by B16 cells. Glycosaminoglycans3H-labeledat their reducing termini or metabolically labeled with [36SJsulfatewere incubated with B16 cell extracts in the absence or presence of D-saccharic acid 1,4-lactone, a potent exo-&glucuronidase inhibitor, and glycosaminoglycan fragmentswere analyzed by high speed gel permeation chromatography. HS isolated from bovine lung, Engelbreth-Holm-Swarm sarcoma, and subendothelial matrix were degraded into fragments of characteristic M,, in contrast to hyaluronic acid, chondroitin 6-suIfate, chondroitin 4-sulfate, dermatansulfate, keratan sulfate, andheparin which were essentially undegraded. Heparin, but not other glycosaminoglycans, inhibited HS degradation. The time dependence of HS degradation into particularM, fragments indicated that HS was cleaved at specific intrachain sites. Ln order to determine specific HS cleavage points, HS prereduced with NaBH4 was incubated with a B 16cell extract andHS fragments were separated. The newly formed reducing termini of HS fragments were then reduced with NaBISHJa,and the fragments hydrolyzed to monosaccharides by trifluoroacetic acid treatment and nitrousacid deamination. Since ‘H-reduced terminal monosaccharides from HS fragmentswere overwhelmingly (>go%)L-gulonic acid, the HS-degrading enzyme responsible is an endoglucuronidase (heparanase)

Metastatic Tumor Cell Heparanase

2284

Methods Cell Extracts-Subconfluent B16 melanoma cells of less than eight passages from an original frozen stock were harvested by treatment for 10 min with 2 mM EDTA in Ca*+,Me-freeDPBS. After suspension into single cells, they were washed twice by brief centrifugation in 0.14 M NaC1, 10 mM Tris-HC1 buffer, pH 7.5, and checked for viability (usually >95%) by trypan blue dye exclusion. Cellswere suspended in chilled 50 mM Tris-HC1 buffer, pH 7.5, containing 0.2% Triton X-100 at a concentration of 6 X lo6 cells/ml. Cell suspensions (1 ml) were sonicated for 20 s a t 4 "C at constantpower using a cell disruptor model W200R (Ultrasonic, Inc., Plainview, NY) equipped with a microtip. The supernatant (approximately 2 mgof protein/ ml) was collected after centrifugation a t 9800 X g for 5 min. Protein contents in the centrifuged extracts were determined by a modification of the Lowry technique (34) to correct for the presence of Triton X-100 in the samples. Glycosaminoglycan Degradation-Purified GAGwasmixed with the centrifuged melanoma cell extract in 0.1 M sodium phosphate buffer (pH 6.0), 0.15 M NaC1,0.2% Triton X-100, 0.05% NaN3 (reaction buffer A). The incubation was carried out a t 37 "C with occasional gentle mixing. The incubation was terminated by chilling the solution to 4 "C and adding of 50%trichloroacetic acid to a final concentration of 5%. After centrifugation at 9800 X g for 5 min, the supernatant was neutralized with 1.0 N NaOH and submitted to further analysis (see below). Gel Chromatography-HS was incubated with B16 cell extracts or heat-inactivated B16 cell extracts in reaction buffer A (pH 6.0), and reactions were terminated as described above. After removal of trichloroacetic acid-insoluble materials, the supernatants were immediately neutralized and passed through small columns of AG 50WX8 (H+form). Acidic fractions were collected on ice, neutralized with 1.0 N pyridine, and lyophilized. The lyophilized samples were dissolved in 1 mlof0.2 M pyridine-acetate buffer, pH 5.0, and applied to a Sephacryl S-200 column (0.9 X 107 cm) previously equilibrated with the same buffer. Elution was performed at a rate of 10 ml/h a t 4 "C; effluent fractions (1 ml) were collected and analyzed for uronic acid by the method of Bitter and Muir (35). Preparation of 3H-labeled GAGS-'H-Labeling of GAGS was performed as follows. One milligram of purified GAG was reduced with 2 mCi of NaB[3H]4(340 mCi/mmol; New England Nuclear) in 0.1 M sodium borate buffer, pH 8.0, at 25 "C for 5 h. After acidification to pH 5 with acetic acid, the mixture was chromatographed on a column (0.9 X 105 cm) of Sephacryl S-200 or S-300 equilibrated with 0.2 M pyridine-acetate buffer, pH 5.0. Individual 3H-labeled GAGS of spe-

cific M, were collected and lyophilized. After dissolving in water, 3Hlabeled GAG was precipitated by the addition of ethanol and NaCl to a final concentration of 80% and 10 mM, respectively. Precipitated GAG was then washed with 80%ethanol and lyophilized to completely remove pyridine. These steps yielded 3H-labeled GAGS with specific radioactivities of 500 to 1100 cpm/pg of GAG. Isohtion of 36S-labeledH S from EHS Sarcoma and BAE Subendothelial Matrix-Primary cultures of minced EHS sarcoma tissues (approximately 3 g) were labeled for 48 h with 25 pCi/ml of Na2[36S] 0, (New England Nuclear) in sulfate-depleted DME medium containing 10% heat-inactivated fetal bovine serum in 10-cm tissue culture dishes. The following steps were carried out at 4 "C except for pronase and chondroitinase ABC digestion. "S-labeled tissues washed with DPBS were frozen and thawed twice and sonicated in 50 mM Tris-HC1buffer, pH 7.5. A 10 times volume of acetone was added and extraction allowed to proceed for 3 h with mixing. At the end of the extraction, insoluble materials were collected by centrifugation, and the sample was re-extracted with acetone and dried completely. The residue was suspended in 20 ml of 1 mg/ml of Pronase (CalbiochemBehring) in 0.15 M NaC1, 7 mM CaCI2, 0.05%NaN3, 10 mM Tris-HC1 buffer, pH 7.5, for 40 h at 37 "C. The mixture was then chilled in an ice bath and mixed with one-fifth volume of 50% trichloroacetic acid. After removal of trichloroacetic acid-insoluble materials by centrifugation for 20 min at 9800 X g, the supernatant was neutralized with 0.1 N NaOH and dialyzed against water. The dialyzed solution was applied to a small AG 50W-X8 (H+ form) column and eluted with water. Acidic fractions were collected, neutralized, and lyophilized. For chondroitinase ABC treatment, lyophilized materials were dissolved in 4 ml of water and then mixed with 1 ml of chondroitinase ABC (Miles, 5 units/ml) in 0.25 M Tris-HC1, 0.3 M sodium acetate, 0.25 M NaCI, and 0.05% bovine serum albumin (Sigma), pH 8.0. After incubation for 18h at 37 "C, the reaction mixture was dialyzedagainst water and subjected to AG 5OW-X8 cation exchange chromatography as described above. Acidic radioactive materials were further fractionated by Sephacryl S-200 chromatography. The major high M, radioactive material was collected and lyophilized. 36S-labeledHS was identified by agarose gel electrophoresis in 1,3-diaminopropane acetate buffer, pH 9.0, according to the method of Dietrich and Dietrich (36). Radioactive materials and standard GAG molecules were detected by toluidine blue staining and autoradiography with Kodak X-Omat AR-5 x-ray film. Purified 35S-labeledHS was resistant to chondroitinase ABC, as well as chondroitinase AC, but was susceptible to nitrous acid deamination (37). %-labeled HS was also prepared from the extracellular matrix of cultured BAE cells. Confluent BAE cell monolayer cultures in IO-cm tissue culture dishes were labeled for 48 h with 25 pCi/ml of Na2[36S] O4in sulfate-depleted DME medium containing 10% heat-inactivated fetal bovine serum, 500 ng/ml of fibroblast growth factor, and 0.1 mM nonessential amino acids. Subendothelial matrix was isolated as described previously (9) and was then digested with 2 ml of Pronase solution as described above. Further steps were performed as described in the method for isolation of HS from EHS sarcoma. The specific radioactivities of "S-labeled HS from EHS sarcoma and BAE subendothelial matrix were 700 and 1500 cpm/pg of hexuronic acid, respectively. Hexuronic acid content was determined by the carbazole-borate method (35) using D-glucuronic acid lactone as the standard. High Speed Gel Permeation Chromatography-High speed gel permeation chromatography was carried out using a high pressure liquid chromatograph system equipped with two sequential columns (0.7 X 75 cm) of Fractogel (Toyopearl) TSK HW-55(S) (MCB, Gibbstown, NJ) as described previously (28). 3H- or =S-labeled GAG was incubated at 37 "C with a B16-BL6 cell extract in 100 pl of reaction buffer A (pH 6.0). The reaction was terminated as described before, and 100 pl of sample solution were delivered into the injection port. Chromatographic elution was performed with 0.2 M NaCl at a flow rate of 1.0 ml/min at 55 "c(28). Effluents were collected each 30 s of elution (0.5-ml volumes), mixed with 3.4ml of Hydrofluor (National Diagnostics, Somerville, NJ), and counted on a Beckman 7500 liquid scintillation counter (Beckman Instruments, Irvine, CA). Preparation ofLabeled MonosaccharideAlcohol Standards-Monosaccharide (1 mg) was reduced with 1 mCi of NaB[3H]4 (170 mCi/ mmol) in 300 pl of 0.01 M NaOH a t 25 "C for 3 h, NaBH. (5 mg) was then added, and thereaction was continued for another 2 h at 25 "C. The reaction mixture was neutralized with 2 M acetic acid and then applied to a small column of AG 5OW-X8 (H+ form) and eluted with


DME/F12 medium supplemented with 10%heat-inactivatedfetal bovine serum (Biocell, Carson, CA), 500 ng/ml of fibroblast growth factor (29), and 0.1 mM nonessential amino acids. The mouse EHS tumor was obtained from Dr. L. A. Liotta (National Cancer Institute, Bethesda, MD) and maintained in C57BL/6 mice by subcutaneous implantation of minced tumor tissue. After 4 weeks ofgrowth tumors (7-8 g) wereexcised and dissected. The tumor tissues were then washed with DPBS (Gibco) and cultured in DME/F12 medium supplemented with 10% heat-inactivated fetal bovine serum (Biocell) and 25 pg/mI of gentamicin (Elkins-Sinn, Cherry Hill, NJ). Glycans-Bovine lung HS was purified according to Cifonelli and Dorfman and Schiller et al. (30, 31), and its average M, (-34,000) was determined by sedimentation equilibrium. Specimens of HA from human umbilical cord, C4S from rock sturgeon notochord, C6S from human umbilical cord, DS, and heparin from porcine mucosal tissue were prepared under a United States Public Health Service National Heart Institute grant and kindly donated by Drs. M. B. Mathews, J. A. Cifonelli, and L. R o d h (University of Chicago, IL). Heparin from porcine intestinal mucosa and bovine lung, and KS from bovine cornea were obtained from Sigma. C6S from shark cartilage and HA from human umbilical cord were obtained from Miles Laboratories (Naperville, IL). Some of these GAGSwere submitted to further purification by gel chromatography on columns of Sephadex G-75, Sephacryl S-200, or S-300 (Pharmacia). Monosialosyl biantennary complex-type glycopeptide UB-I-b was prepared from thyroglobulin (Sigma) according to Yamamoto et al. (32). Tri-N-acetylchitotriose was prepared from crab shell chitin according to Rupley (33). D-Galactose,D-XylOSe, and 2-deoxy-~-glucose were purchased from Calbiochem-Behring.N-Acetyl-D-glucosamine,D-glucosaminehydrochloride, SAL, and D-glucuronic acid lactone were purchased from Sigma. D-Glucuronic acid was purchased from K and K Chemicals (Plainview, NY). L-Iduronic acid was isolated from heparin.

Metastatic Tumor Cell Heparanase

RESULTS

Gel Chromatographic Analysis of HS and Its Fragments Produced b y B16 Cell Extracts-To determine the relative

Elution profiles of purified bovine lung HS showed a sharp single peak (Fig. 1A). In the presence of B16 cell extracts the original HS peak decreased in amount and peaks of lower M, appeared. After 6 h of incubation, degradation components of M, approximately 22,000, 12,000, and 4,000 appeared, and free hexuronic acids were detected (Fig. IA). When the incubation was performed in the presence of SAL (20 mM), the

A

E

0.3

c

d

e

I

1

i

m In


water, except for 3H-labeledD-ghlcosaminito~,which was eluted from the AG 50W-X8 column with 1 M ammonia. Boric acid was removed by repeated evaporation with methanol, and radioactive contaminants were removed using descending paper chromatography (Whatman No. 1)in I-butanol-pyridine-water (6:4:3) (38). Analysis of Reducing Terminal Saccharides of HS Degradation Fragments-Purified HS (5 mg) from bovine lung was reduced in 2 ml of 0.5 M borohydride, 0.1 M borate buffer, pH 8.0, at 22 "C for 3 h. Excess borohydride was destroyed by acidification to pH 5.0 with 2 M acetic acid; the mixture was applied to a Sephacryl S-200 column (0.9 X 107 cm) equilibrated with 0.2 M pyridine-acetate buffer, pH 5.0, and itwas then eluted with the same buffer. Eluent was monitored by measuring hexuronic acid according to Bitter and Muir (35). Reduced HS fractions were collected, lyophilized, and then washed with ethanol to remove pyridine. Reduced HS (2 mg) was incubated at 37 "C for 12 h with a B16-BL6 cell extract (1 mg of protein) in 2 ml of reaction buffer A (pH 6.0) in the presence of 20 mM D-saccharic acid 1,4-lactone, a lysosomal @-glucuronidaseinhibitor (39). The reaction was terminated by chilling to 4 "C, and 220 p1 of 50% trichloroacetic acid were added. After the samples were centrifuged at 9800 X g for 5 min at 4 "C, the supernatantswere neutralized with 1 N NaOH, and ethanol and barium acetate were added to final concentrations of 80 and 0.376, respectively. The mixtures were left a t 4 "C for 20 h, and theprecipitates were collected by centrifugation as before. Precipitates were dissolved in water and applied to a small column ofAG 50W-X8 (H+ form) eluting with water. The passthrough fractions were lyophilized and chromatographed on Sephacryl S-200 as described above. Reducing terminal saccharide residues of HS degradation products were analyzed as follows. Fractionated HS fragments were reduced with NaB[3H]r as described in the method for preparation of 'Hlabeled monosaccharide alcohols. The reaction mixtures were neutralized with acetic acid and chromatographed on Bio-Gel P-10 columns (0.6 X 20 cm) with water to isolate 3H-labeled products. Degradation of 3H-reduced HS fragments to monosaccharides was performed by one of the following methods: 1) acid hydrolysis in 4 N HCI at 100 "C for 8 h 2) acid hydrolysis in 2 M trifluoroacetic acid, deamination in 3.9 M NaN02-0.28 M acetic acid, and further acid hydrolysis in 2 M trifluoroacetic acid according to the method of H66k et al. (40). After repeated evaporation of hydrolyzed samples in the presence of water, the hydrolysates were dissolved in 2 ml of 0.5 M Tris-HC1 buffer, pH 8.0, and were left a t 25 "C for 24 h to convert aldonic acid lactones into free acids (40). These samples were chromatographed on Bio-Gel P-2 columns (0.6 X 90 cm) with water. Monosacchaide fractions were collected and applied to DEAE-Sephacel columns (0.5 X 30 cm). The columns were successivelyeluted with 50 ml of water and 50 ml of 0.4 M pyridine-acetate buffer, pH 3.0. Pass-through fractions were repeatedly subjected to DEAE-Sephacel chromatography after treatment with 0.25 M ammonia to recover quantitatively hexonic acids in the acidic fractions. Nonacidic monosaccharides were analyzed by descending paper chromatography (Whatman No. 1) in 1-butanol/pyridine/water(6:4:3) (38) and by high voltage paper electrophoresis (Savant Instruments, Inc., Hicksville, NY) in 0.06 M borate buffer, pH 8.9 (41). Acidic monosaccharides (aldonic acids) were identified by paper chromatography of their corresponding aldono-1,4-lactones (42) in t-amyl alcohol/isopropyl alcohol/water (4:1:2) (43). 3H-labeled monosaccharides were detected by radioactivity measurement, and reduced and nonreduced monosaccharides were detected by silver nitrate staining after short-term periodate oxidation according to themethod of Yamada et a/. (44).

2285

20

30

40

50 60

70

80

Elution Volume ( ml )

FIG. 1. Sephacryl 5-200 gel chromatography of HS incubated with B16-BL6 melanoma cell extract in the absence or

TumorMetastatic

2206

Cell Heparanase

m

0

At 3 h the peaks of M,= -15,000, -10,000, and -5,400 were demonstrable, and a fragment of M, 5,400 accumulated after 6 h of incubation. These profiles are much simpler than the gel chromatographic profiles based on total hexuronic acid contents and strongly suggest that B16 heparanase cleaves bovine lung HS at a minimum of five intrachain sites. We could not detect any preferences in the cleavage of heparan sulfate at any of these intrachain sites. The following other GAGs with 3H-labeled reducing terminal saccharides were also examined HA from human umbilical cord (M, 230,000), C4Sfrom rock sturgeon notochord (Mr 12,000), C6S from shark cartilage (MI SO,OOO), DS from porcine mucosal tissue (Mr 27,000), KS from bovine cornea (M, 14,000),heparin from bovine lung ( M , 15,000), and heparin from porcine intestinal mucosa (MI 11,000). Incubation with aB16-BL6 cell extract was carried out under the same conditions as the HS degradation assay. Elution profiles of these GAGs (except bovine lung heparin) and some of their degradation products are shown in Fig. 3. Per cent of degradation of these GAGs calculated from the decrease in

-

-

-

-

-

-

-

HEPARIN

.. .. ,

*.

. *

100 10

.. . '*.

1

**,

c * "

20

30

40

EHS-HS

.* .*

20

A .

40

30

BAE-HS

.* .**

.* .. .:.'

A '

L

. . *

-*

.

*" .a'

""

40 20 30 Retention tlrne ( rnln )

FIG. 2. High speed gel permeation chromatography of HS and its fragments produced by B16 heparanase. A, logarithmic plot of M , uersus the retention times of standard glycans separated on two sequential columns of Fractogel-TSK HW-556). Standard glycans are: a, HA from human umbilical cord (M, 230,000); b, C6S from shark cartilage (M, 60,000); c, HS from bovine lung (M, 34,000); d , DS from porcine mucosal tissue (Mr 27,000); e, C4S from notochord of rock sturgeon ( M , 12,000); f, heparin from porcine mucosal tissue (M, 11,000); g, monosialosyl biantennary complex-type glycopeptide from porcine thyroglobulin (Mr 2,190); h, tri-N-acetylchitotoriose (M, 627); i, N-acetyh-ghcosamine ( M , 221). B, elution profiles of 3H-labeled HS (0)and its degradation fragments produced by B16 heparanase (0).3H-labeled HS (5 rg, 2,500 cpm) was incubated at 37 "C with a B16-BL6 cell extract (40 pg of protein) in reaction buffer A (pH 6.0) in the presence of 20 mM SAL. 3H-labeled HS and itsdegradation fragments were fractionated by high speed gel permeation chromatography. Arrows a-i indicate the eluting positions of the standard glycans.

-

-

-

-

-

-

-

-

20

30

40 RETENTION TIME

20

30

40

( MIN )

FIG. 3. High speed gel permeation chromatographic analysis of GAGs incubated with B16 cell extracts. 3H- or 35S-labeled GAG (10 pg) was incubated with a B16-BL6 cell extract (60 pgof protein) in reaction buffer A (pH 6.0) for 6 h at 37 "C in the absence or presence of 20 mM SAL. Incubation products were analyzed by high speed gel permeation chromatography. HA, 3H-labeledHA from human umbilical cord DS, 3H-Iabeled DS from porcine mucosal tissue; KS, 3H-labeled KS from bovine cornea; C6S, 3H-labeled C6S from shark cartilage; C4S, 3H-labeled C4S from rock sturgeon; Heparin, 3H-labeled heparin from porcine intestinal mucosa; EHS-HS, [%]O~labeled HSfrom EHS sarcoma; BAE-HS, [36S]04-labeled HS from BAE subendothelial matrix. A , GAG incubated with a heatinactivated B16-BL6 cell extract; B , GAG incubated with a B16-BL6 cell extract in the presence of 20 mM SAL; C, GAG incubated with a B16-BL6 cell extract without SAL.


intermediate M,components remained, while the appearance of low M,components decreased (Fig. 1B). At the monosaccharide-eluting position, SAL was eluted quantitatively as indicated by carbazole-borate reaction. When the materials in themonosaccharide fractions were analyzed by paper chromatography (38, 43) and high voltage paper electrophoresis (41), neither glucuronic acid nor N-acetylglucosamine was detected. Therefore, the degradation of HS to intermediate M,fragments was not significantly affected by the exoglycosidase inhibitor, indicating that HS degradation was mainly due to endoglycosidase(s). Higher concentrations of SAL (40 mM) markedly inhibited HS degradation, suggesting that HSdegrading endoglycosidases (heparanase) may be sensitive to high SAL concentrations. Analysis of Cleavage Products from GAGS Labeled with 3H at Their Reducing Ends-HS with 3H-labeled reducing terminal saccharides were incubated with B16-BL6 celI extracts in the presence of SAL, and the HS degradation products were analyzed by high speed gel permeation chromatography (28). In this experiment, only fragments having original reducing terminal residues could be identified. High speed gel permeation chromatography of HS degradation products and standard glycans are shown in Fig. 2. After 1 h of incubation with a B16-BL6 cell extract in the presence of SAL, the amount of glycan in the original HS peak decreased, while fragments of M,= -22,000, -15,000, and -10,000, appeared.

Metastatic Tumor Cell Heparanase TABLE I High speed gel permeation chromatographic studyof GAG degradation by a B16 melanoma cell extract GAG degrada-

"_

Inhibition of HS degrada

t.inn'

GAG

OmM

20mM

SAL

SAL

HS (bovine lung, M ,

95.2

88.5

21.3

-34,000)' HS (EHS sarcoma, M,

92.3

90.5

NT"

tion*

%

- 70,000)d

-

-

-

-

-

-

-

-

labeled HS isolated from EHS sarcoma was degraded at high rates duringan incubation with a B16-BL6 cell extract in the presence of SAL (Fig. 3 and Table I), and characteristic M, degradation peaks appeared (average M,= -24,000, -14,000, -9000, and -5600) indicating that EHS sarcoma HS was discontinuously fragmented by B16 heparanase. [35S]0d-labeled HS from BAE subendothelial matrix was also degraded at high rates (Fig. 3 and Table I); however, this HS appeared to be fragmented into larger M, molecules (average M, 8000) than the degradation products of the other HS molecules. In the absence of SAL, HS fragments were further degraded and broad fractionation peaksappeared, suggesting the action of exoglycosidases. Effects of GAGS on H S Degradation by B16 HeparameThe interactionsof B16 heparanase with various GAGs were examined using a heparanaseinhibition assay. GAG was added to a B16-BL6 cell extract incubation mixture containing an equal amount (dry weight) of 3H-labeled HS, and the effects of GAGs on HS fragmentation were determined by high speed gel permeation chromatography. When equal amounts of HS and 3H-labeled HS were present, the rate of 3H-labeled HS degradation decreased slightly below that of 3H-labeled HS alone (Fig. 4 and Table I). In contrast, the addition of porcine intestinal mucosa heparin almost completely inhibited the appearance of intermediate M , HS fragments (Fig. 4). Bovine lung heparin also showed this inhibitory effect (Table I). These results indicated that B16 heparanase can bind but not cleave the major GAG components of heparin. Other GAGs (HA, C6S, C4S, DS, and KS) tested did

-

I

I

I

I

I

I 1

'

area of the high M, half of the GAG peak is listed in TableI. In the presence of SAL, none of the GAGs was detectably affected after prolonged incubation. For example, the elution profile of porcine intestinal mucosa 3H-labeled heparin incubated with a B16-BL6 cell extract in the presence of SAL showed no alterations in M , from that of the [3H]heparin control(Fig. 3). In the absence of SAL heparin was only partially degraded. Partial degradation of C6S was also observed in the absence of SAL (Table I), indicating that B16 melanoma exoglycosidases may be involved. The partial degradation of GAGs in theabsence of SAL may be due to GAG molecular heterogeneity. Degradation of P5S]04-lnbeled HS isolated from EHS sarcoma or BAE Subendothelial Matrix-To examine the action of B16 heparanase on HS prepared from various tissues, we have used PYS carcinoma (28), EHS sarcoma, and BAE and BCE subendothelial matrix.* EHS sarcoma synthesized very large M , [35SJ0,-labeled HS (average M , 70,000) during short-term culture (Fig. 3). HS from BAE subendothelial matrix was M, 24,000; however, cell-associated HS from BAE cells was much smaller (average M, 8000). [36S]04-

-

-

-

'2. Wang, T. Irimura, M. Nakajima, P. N. Belloni, and G. L. Nicolson, Eur. J . Biochern., submitted for publication.

I ."...D

2**..-

20

. 30

LO

Retention time ( min )

FIG. 4. Effect of heparin on HS degradation by B10 heparanase. Ten micrograms of 3H-labeled HS from bovine lung was incubated with a B16-BL6 cell extract (60 pg of protein) in reaction buffer A (pH 6.0) containing 20 mM SAL for 6 h a t 37 'C in the absence or presence of heparin from porcine intestinal mucosa. After termination of the incubation, samples were analyzed by high speed gel permeation chromatography. Elution profiles are: A, t3H]HS incubated with a heat-inactivated cell extract; B, [3H]HS incubated with a cell extract in the presence of 10 pgof heparin; C, [3H]HS incubated with a cell extract in the presence of an additional 10 pg of unlabeled HS; D, [3H]HS incubated with a B16-BL6 cell extract. Arrows a, b, c, f ,g,and h indicate the elution positions of the standard glycans indicated in Fig. 2.


93.5 NT 96.5 elialmatrix, M, 24,000)d 83.6 12.3 15.6 Heparin (bovine lung, M, 15,000)' 7.1 85.3 12.0 Heparin (porcine intestinal mucosa, M, 11,000)'