Preparation of GBM Heparan Sulfate Proteoglycan-Basement membranes ..... The arrows indicate the elution of N-acetylated hydrazine-released N-linked car-.
Vol. 262, No. 14, Issue of May 15, pp. 6893-6898 1987 Printed in C.S.A.
THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1987 by The American Society of Biological Chemists, Inc.
Selective Deglycosylation of the Heparan SulfateProteoglycan of Bovine Glomerular Basement Membraneand Identification of the Core Protein* (Received for publication, December 5, 1986)
Albert S. B. Edge$ and RobertG . Spirofj From the Departments of Biological Chemistry and Medicine, Harvard Medical Schooland the Elliott P. Joslin Research Laboratory, Boston, Massachusetts 02215
(1,2 ) , is an integratedmulticomThe heparan sulfateproteoglycan of the bovine glo- the renal filtration apparatus merular basement membrane (M, = 200,000,30%car- ponent structure consisting of collagen,proteoglycan, and is to be defined bohydrate by weight) hasbeen deglycosylated by var- several glycoproteins.Its permeability believed ious chemical and enzymatic procedures to identify theby pore size and electrostatic charge, and the latter property core protein and provide information aboutNthe and has beensuggested to be due primarily to anionic groups 0-linked saccharide units. Heparitinase digestion of present on protein-linked heparan sulfate chains(3). the proteoglycan reduced its M, to 143,000, consistent Previous studies carried out in our laboratory have indiwith the removal of its fourglycosaminoglycan chains cated that mostof the hexuronic acid of bovine GBM occurs with the exception of short segments adjacent to the in a heparan sulfate proteoglycan with a molecular weight of carbohydrate-protein linkageregion, whereas nitrous 200,000 which contains four glycosaminoglycan chains (M, = acid treatment brought about a smaller reduction in 14,000) as well as several N-linked and small 0-linked carsize (to M, = 168,000) which was shown to be due to bohydrate units(4).However, the major part of this glycoconthe resistance of the internal portion of the heparan sulfate polymer to this reagent. Incubation of the he- jugate (70% by weight) was found to consist of polypeptide paritinase-digested proteoglycan with peptide N-gly- (4);this portion of the molecule is of considerable interest cosidase F decreased its M, by about 8,000 and liber- since it is likely that in addition to providing sites for the ated oligosaccharides which were primarily acidic in attachment of the various saccharide chains it may play an nature; since endo-0-N-acetylglucosaminidaseH did important role in the interaction of the proteoglycan with macromolecules and contribute not bring about any saccharide release, it appears that otherbasementmembrane the N-linked carbohydrate units (three per molecule) substantially to its antigenicity. In the present investigation, by applying enzymatic and occur exclusively as the complex type. Treatment of the proteoglycan with trifluorometh- chemical deglycosylation procedures to thebovine GBM proanesulfonic acid, a reagent which cleaves all sacchar- teoglycan, it hasbeen possibleto selectively remove or shorten ide units, yielded the core proteinwhich migrated as a various carbohydrate chains, and this hasled to the identifisingle discrete band (M. = 128,000)on polyacrylamide cation of the core protein. Moreover, additional information gel electrophoresis. Although the native and hepariti- about the saccharide units was obtained from a study of the nase-treated proteoglycan reacted with concanavalin deglycosylation products aswell as from an assessmentof the A and Bandeiraea simplicifoliaI, the core protein had interaction of native andmodified proteoglycan with concanno affinity for these lectins, and this loss of reactivity avalin A and Bandeiraea simplicifolia I* lectins. An evaluation can be attributed to the removal of the N- and small 0- of the immunological reactivity of the deglycosylated proteolinked saccharides. However, the immunoreactivityof glycan indicated thatpolyclonal antibodies raised against the the deglycosylated proteinwithantiserumdirected its polypeptide native molecule are primarily directed toward against the intact proteoglycan was toa large measure (80%) preserved, suggesting that the polyclonal re- portion. sponse to this glomerular basement membrane glycoEXPERIMENTALPROCEDURES conjugate is primarily directed against determinants Preparation of GBM Heparan Sulfate Proteoglycan-Basement on the polypeptide portion.
The GBM,’ which functions as the primary constituent of * This work was supported by Grant AM 17325 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. $Recipient of a Capps Scholarship in Diabetes from Harvard Medical School. To whom correspondence should be addressed Elliott P. Joslin Research Laboratory, 1 Joslin Place, Boston, MA 02215. The abbreviations used are: GBM, glomerular basement membrane; TFMS, trifluoromethanesulfonic acid; endo H, endo-P-N-acetylglucosaminidase H; SDS, sodium dodecyl sulfate; PBS, phosphatebuffered saline.
membranes obtained from bovine renal glomeruli were extracted with 4 M guanidine HC1 as previously described (4),and the solubilized proteoglycan was purified by DEAE-cellulose chromatography (4) employing a modified sodium chloride elution gradient (2). Preparation of Heparan Sulfate Chains-After treatment of the proteoglycan (2 mg) with 0.2mlof 0.1 N NaOH containing 1 M NaB3H4(Du Pont-NewEngland Nuclear, adjusted to 110 mCi/mmol) for 72 h a t 37 “C, the reduced radiolabeled glycosaminoglycan chains were purified by Bio-Gel filtration and DEAE-cellulose chromatography as previously reported (4). Radiolabeling of Proteoglycan-Reductive alkylation of the proteoglycan was performed with [14C]formaldehyde(Du Pont-New England Nuclear, 52 mCi/mmol) as described (4) and resulted in a product with a specific activity of 9.1 X IO6 dpm/mg. TFMS Treatment of Proteoglycan-Deglycosylation of the “Clabeled proteoglycan (1.2 X lo6 dpm) as well as this unlabeled glyco-
6893
This lectin is also known as Griffonia simplicifoliaI.
6894
Deglycosylation of Glomerular Basement Membrane Proteoglycan
conjugate (0.5 mg) was accomplished in 100 81 of TFMS-anisole (2:1, v/v) reagent (5) under nitrogen for 3 h a t 0 "C. Neutralization and ether extraction were carried out aspreviously reported (5), followed by dialysis against 20 mM ammonium bicarbonate; the yield after these procedures was 60% of the startingmaterial. Nitrous Acid Treatment of Proteoglycan and Free HeparanSulfate Chains-Unlabeled (0.5 mg) and "C-labeled (1.2 X lo5 dpm) proteoglycan as well as theNaB3H4-reducedglycosaminoglycanchains (4 X lo5 dpm) were treated with nitrous acid reagent as previously described (6). After this procedure the proteoglycan samples were dried and extracted three times with 80% ethanol, whereas those containing the free heparan sulfate chains were applied to a column (1.8 X 127 cm) of Bio-Gel P-10(100-200 mesh) equilibrated with 0.1 M pyridine acetate, pH 5.0, a t a flow rate of 12 ml/h. Heparitinase and Heparinuse Digestions-Radiolabeled and unlabeled proteoglycan (1.8 X lo5 dpm or 0.5 mg, respectively) as well as NaB3H4-reducedheparan sulfate chains(4 X lo5dpm) were incubated in 100 pl of 0.05 M Tris-HC1 buffer, pH 7.2, containing 0.02 M calcium acetate for 16 h at 37 "C with either 10 milliunits of heparitinase or heparinase from Flavobacterium heparinum (Miles Laboratories) in the presence of toluene; in the case of the proteoglycan, 1 mM phenylmethylsulfonyl fluoride and 10 pg each of antipain, leupeptin, phosphoramidone, elastatinase, pepstatin, amastatin, and bestatin were included in the incubation to serve as protease inhibitors (7). The digestions were terminated by heating a t 100 "C for 2 min; proteoglycan was desalted on a column of Bio-Gel P-2 (1 X 25 cm) equilibrated in 0.05 M ammonium bicarbonate, whereas samples containing the heparan sulfate chains were fractionated on Bio-Gel P-10. Peptide N-Glycosidase F and Endo H Digestwns-Radiolabeled proteoglycan (2 X 10' dpm),both before and afterheparitinase treatment, was digested with 0.26 units peptide N-glycosidase F from Flavobacterium meningosepticum (Genzyme, N-glycanase) in 30 pl of 0.1 M sodium phosphate, pH 8.6, containing 0.17% SDS, 0.33% 2mercaptoethanol, 6 mM EDTA, 0.6% Nonidet P-40, and 2 mM phenylmethylsulfonyl fluoride or 10 milliunits of endo H (Streptomyces griseus, Miles Laboratories) in 100 pl of 0.15 M sodium citrate, pH 5.2, containing 0.1% bovine serum albumin and either 0.05%SDS or 0.5 M sodium thiocyanate (8). Incubations were carried out a t 37 "C for 16 h in the presence of toluene; subsequently, the samples were treated a t 100 "C for 2 min, and aliquots were removed for examination by polyacrylamide gel electrophoresis. In order to characterize the oligosaccharides released by enzyme action, unlabeled heparitinase-digested proteoglycan (50 pg) was incubated with the endoglycosidasesunder the conditions already specified. After removal of salt by passage of the peptide N-glycosidase F digest through a column of Bio-Gel P-2 (equilibrated in 0.1 M pyridine acetate, pH 5.0) and of the endo H-treated sample through coupled columns of Dowex 50-X2 (200-400 mesh, H+form) and Dowex 1-X2 (200-400 mesh, acetate form), the liberated oligosaccharides were reduced with NaB3H4(1mCi, 7.5 Ci/mmol) in 100 pl of 0.2 M sodium borate buffer, pH 9.0, for 4 h at room temperature. The reaction was terminated by addition of acetic acid followedby passage of the samples through Dowex 50-X4 (H+ form) and volatilization of boric acid as methyl borate. After descending paper chromatography for 10 h in pyridine/ethyl acetate/water/acetic acid (5:5:3:1), the oligosaccharides which remained a t the origin under theseconditions (9) were eluted with water for further examination. The reduced endo H products were subjected to thin layer chromatography on Silica Gel 60-coated plates in l-propanol/acetic acid/H20 (3:3:2) as previously described (9), whereas the oligosaccharides from peptide N-glycosidase F digestion were filtered on a calibrated column (1.8X 130 cm) of Bio-Gel P-4 (400 mesh) in 0.1 M pyridine acetate, pH 5.0, followed by chromatography on a DEAE-cellulose (DE52, microgranular) column (0.7 X 4 cm) with a linear gradient (80 ml) ranging from 5 mM to 1 M pyridine formate, pH 5.0. Solid-phase Lectin Binding Assay-The interaction of native and deglycosylated proteoglycan with concanavalin A (Miles) and B. simplicifoliaI (Vector) was measured by coating microtiterwells with the protein (10-100 ng) to be assayed as previously described (10). The lectin was then added to the washed wells as 200 pl of a 2 pg/ml solution in PBS(0.05 M sodium phosphate buffer, pH 7.4, containing 0.15 M NaCl) containing 0.05%Tween 20 and 0.1% bovine serum albumin and allowed to interact overnight at 4 "C. After five washes with PBS/Tween/bovine serum albumin, 100 pl of the appropriate 1:lOO diluted rabbit anti-lectin antiserum (E-Y Laboratories) was added and kept in the wells at room temperature for 2 h. The wells were again washed with PBS/Tween/bovine serum albumin, and this
was followed by the addition of 100 pl (1 X 10' cpm, 3-5 ng) of labeled protein A radioiodinated by the chloramine-T procedure (11); after a 2-h incubation a t room temperature, the wells were washed six times and assayed for radioactivity in a gamma counter. Antiserum Preparation and Assay-The preparation of antiserum (12) against the native proteoglycan from bovine GBM (4) has previously been reported (2). The specificity and titer of the antiserum were determined by a solid-phase radioimmunoassay (2). Polyacrylamide Gel Electrophoresis and Immunoblot AnulysisElectrophoresis was carried outinSDSin the buffer system of Laemmli (13) utilizing vertical polyacrylamide slab gels(1.5mm thick) with a linear 3-13% acrylamide gradient and a 2.5% stacking gel. The components were detected by staining with Coomassie Blue or by fluorography. For immunological identification the proteins were transferred onto a nitrocellulose sheet (14) which was incubated with antiserum (1:lOOO dilution) and after washing exposed to 1251-labeled protein A as previously described (2); the radioactive bands were detected by autoradiography. Protein Determinution-Protein was estimated by the procedure of Bradford (15) using bovine serum albumin as a standard. Radioactivity Measurements-Liquid scintillation counting was performed with Ultrafluor (National Diagnostics) in a Beckman LS 7500 instrument; a Model1290 Gamma Trac was employed for gamma counting. Radioactive components on thin layer plates, nitrocellulose sheets, and electrophoretic gels weredetected by autoradiography or fluorography a t -70 "C after treatment with ENHANCE (Du Pont-New England Nuclear) using X-Omat AR film (Eastman Kodak). RESULTS
Identification of the Bovine GBM Heparan Sulfate Proteoglycan CoreProtein-Treatment of the '*C-labeled proteoglycan with TFMS resulted in a reduction of its apparentmolecular weight from 200,000 to 128,000 as determined by SDSpolyacrylamide gel electrophoresis (Fig. 1). This decrease in mass was consistent with the removal of the various carbohydrate units which together have been reported to account for 30% of the molecule's weight (4). Moreover, the pronounced sharpening of the electrophoretic band brought about by the TFMS treatment (Fig. 1) indicated that the broad
Origin
-
-
200-
116-
I) a
66 -
4529
-
FIG. 1. Comparison of the effects of various saccharide removal procedures on the migration of GBM heparan sulfate proteoglycan during polyacrylamide gel electrophoresis in SDS.The "C-labeled proteoglycan (9,000 dpm) was electrophoresed directly (Native) or after treatment with nitrous acid (HN02),heparitinase (HSase), or TFMS as described under "Experimental Procedures.'' The components were visualized by fluorography; the designated molecular weight markers weremouse laminin a-subunit (200,000), Escherichia coli @-galactosidase(116,000), bovine serum albumin (66,000), hen ovalbumin (45,000), and bovine erythrocyte carbonic anhydrase (29,000).
Deglycosylation of Glomerular Basement Membrane Proteoglycan appearance of the native proteoglycan on the polyacrylamide gel is due to variability in its saccharide chains. Comparison of the Effect of Nitrous Acid and Heparitinuse Treatment on the Proteoglycan and Its Glycosaminoglycan Chains-Nitrous acid degradation of the native proteoglycan reduced its apparent molecular weight to approximately 168,000,whereas heparitinase digestion brought about a more substantial change with the appearance of a component with an M , of 143,000(Fig. 1). The product of this enzymatic treatment migrated on electrophoresis to a position intermediate between that of the nitrous acid-degraded and the TFMS-deglycosylated proteoglycan, indicating that the four ( M , = 14,000) heparan sulfate chains (4) had been substantially shortened. However, incontrast to the pronounced effect brought about by heparitinase, digestion of the proteoglycan with heparinase did not result in a detectable change in its electrophoretic migration (data not shown). In order to correlate the molecular weight changes brought about by nitrous acid and heparitinase on the intact proteoglycan to cleavages occurring within the heparan sulfate chains, the effect of both of these treatments on the isolated NaB3H4-reducedglycosaminoglycans wasevaluated. As anticipated from a previous study (4), these chains, which terminate in radiolabeled [3H]xylitol, were converted by nitrous acid treatment from material which eluted (Kev= 0.07) near the void volumeof a Bio-Gel P-10 column to fragments which were included in such a column; most of the radioactivity (73%) emerged as a major peak (Kav = 0.35)) although a spectrum of smaller components was also evident (Fig. 2). Digestion of the glycosaminoglycan chains with heparitinase effected a more pronounced reduction in molecular weight, and a peak which represented the short stubs from the reducing end eluted with a Kevof 0.64 (Fig. 2). The heparan sulfate chains, like the intactproteoglycan, were found to be resistant to theaction of heparinase (data notshown). Release of N-Linked Carbohydrate Units from Proteoglycan by Peptide N-glycosidase F Treatment-Digestion of the heparitinase-treated proteoglycan with peptide N-glycosidase F, an enzyme which cleavesboth polymannose and andcomplex N-linked saccharide units (16), resulted in a further reduction
6895
in its molecular weight (M, = 135,000). The product of this treatment, although migrating faster than the proteoglycan digested only with heparitinase, still moved more slowlythan the TFMS-deglycosylated molecule (Fig. 3).Incubation of the native proteoglycan with peptide N-glycosidase F brought about no detectable change in molecular weight (data not shown). In contrast to peptide N-glycosidase F, endo H digestion of the heparitinase-treated proteoglycan failed to bring about any change in electrophoretic migration when either sodium thiocyanate or SDS wereemployed as denaturants. Furthermore, no released polymannose oligosaccharides (Man9GlcNActo Man5GlcNAc) wereevident upon thin layer chromatographic examination of NaB3H4-reducedproducts under conditions in which less than 0.03 nmol of oligosaccharide/nmol of protein were clearly evident in concurrent digestions of calf thyroglobulin (17); even endo H treatment of pronase-digested proteoglycan (18), to eliminate potential steric interference, failed to result in a detectable release of carbohydrate units. The oligosaccharides released by peptide N-glycosidase F couldberesolved after NaB3H4 reduction by Bio-Gel P-4 filtration into three peaks with average molecular weightsof 4700,3100, and 2300, respectively,whichwere present in molar ratios of 0.5:1.00.4 (data not shown). When the components in the peak of intermediate size (Mr = 3100) were further fractionated on DEAE-cellulose, charge heterogeneity comparable to that observed in fetuin N-linked saccharide units (19) was evident (Fig. 4). Thin layer chromatography after re-N-acetylation of the hexosamines released by acid hydrolysis of the peptide N-glycosidase F-liberated oligosaccharides indicated, as anticipated, that radiolabeled N-acetylglucosaminitol was in the terminal position (data notshown). Effect of Deglycosylation onthe Interaction of Proteoglycan with kctins-With the use of a sensitive solid-phase assay which employs anti-lectin sera and radiolabeled protein A to
200 11666-
45
-
29 -
I
I
1
20
30
40
1 50
60
TUBE NUMBER 1 6 m l )
FIG. 2. Comparison of the effects of nitrous acid and heparFIG. 3. Effect of peptide N-glycosidase F treatment on the itinase treatment on the heparan sulfate chains of the GBM proteoglycan as determined byfiltration on Bio-Gel P- 10.The migration of GBM heparan sulfate proteoglycan during polNaB3H,-reduced glycosaminoglycan (4 X lo5 dpm) was applied to the yacrylamide gel electrophoresis in SDS comparedtoother column (1.8 X 127 cm) before (- - -) and after treatment with nitrous deglycosylation procedures.The 14C-labeled proteoglycan (9000 dpm) was applied to the gel after heparitinase (HSase), TFMS, or acid (0)or heparitinase (0).The column was eluted with 0.1 M pyridine acetate, pH 5.0, a t a flow rate of 12 ml/h. V , and V, refer, heparitinase plus peptide N-glycosidase F (HSase + N-Glyase) treatrespectively, to the void and total volumes of the column; KS desig- ment as described under “Experimental Procedures.” The componates the elution position of keratan sulfateglycopeptide (average M, nents were visualized by fluorography; the molecular weightmarkers were the same as in Fig. 1. = 10,000).
Deglycosylation of Glomerular Basement MembraneProteoglycan 1 2 3
4
ATlVE
bJ'
Con A
BS-I
FIG. 6. Effect of saccharide removal on the reactivity of the GBM proteoglycan with concanavalin A (Con A ) and B. aim, plicifolia I (BS-I). The intact (NATIVE), heparitinase-digested 0 10 20 30 (HSase),and TFMS-treatedproteoglycan (50 ng of each) were coated TUBE NUMBER l2rnll onto microtiter wells and assayed for lectin binding by the method FIG. 4. Chromatography on DEAE-cellulose of NaB3H4-re- described under "Experimental Procedures." duced oligosaccharides released from GBM proteoglycan by 1 peptide N-glycosidase F. The major saccharide peak (average M , = 3,100) obtained by filtration on Bio-Gel P-4 was applied (10,000 dpm) to thecolumn equilibrated with 5 mM pyridine formate, pH 5.0, and after awash with this buffer elution was carried out with a linear L gradient as described under "Experimental Procedures." The arrows indicate the elution of N-acetylated hydrazine-released N-linked carL bohydrate units from fetuin containing, respectively, 1, 2, 3, and 4 sialic acid residues.
. coNAr /
6l
4 t
0
I
1
I
I
5
IO
15
20
ANTIGEN ( n g )
FIG. 7. Comparison of the reactivity of native and deglycosylated proteoglycan with anti-bovine GBM heparan sulfate proteoglycan serum. Native, heparitinase-digested (HSase), and TFMS-treated GBM proteoglycan were assayed in increasing amounts by solid-phase radioimmunoassay using '251-labeledprotein A as described under "Experimental Procedures;" the serum was employed at 1:lOOO dilution.
-
-
IV
a 0 a a
ov
BS-I
measure lectin-glycoconjugateinteraction, the binding of concanavalin A and B. simplicifolia I to the GBM proteoglycan could be demonstrated (Fig. 5 ) . The specificity of the assay was established by noting that the appropriate methylglycoside inhibited lectin binding to proteoglycan and thata number of standard glycoproteins, namely ovalbumin (20), fetuin (21), laminin (22), and thyroid GP-1 (23), reacted with the concanavalin A and B. simplicifolia I as would be anticipated I 1 I I from the nature of their carbohydrate units (Fig. 5). 0 20 40 60 00 100 The lectin-binding assay provided a sensitive tool for assessing the removal of saccharides by deglycosylation procePROTEIN ( n g ) dures; the binding of both concanavalin A and B. simplicifolia FIG. 5. Evaluation of the reactivity of the GBM proteogly- I to the proteoglycan was decreased by more than 95% after can with lectins as determined by solid-phase immunoassay. The binding of concanavalin A (ConA ) and B. simplicifolia I (BS-I) TFMS treatment; in contrast, enzymatic removal of heparan to micro-wells coated with increasing amounts of proteoglycan (PG) sulfate chains did not significantly alter the extent of these as well as standard glycoproteins, ovalbumin (OV), laminin ( L M ) , interactions (Fig. 6). 0
2 -
I
I
I
fetuin (Fet), and thyroid GP-1 (GP-1)was determined by an assay described under "Experimental Procedures" in which anti-lectin serum followed by '251-labeledprotein A are employed. To test the specificity of the lectin interaction, the proteoglycan was incubated with Con A and BS-I in the presence of 100 mM methyl-a-D-mannoside (PC CH3-Mun)and 100 mM methyl-a-D-galactoside (PG CH3-Gal),respectively.
+
+
Immunochemical Properties of Proteoglycan after Deglycosylation-As indicated by solid-phase radioimmunoassay, de-
glycosylation had little effect on the interaction of the proteoglycan with an antiserum raised against the native molecule (Fig. 7). The heparitinase-digested proteoglycan bound antibody to thesame extent asthe intactglycoconjugate, whereas
Deglycosylation of Glomerular Basement Membrane Proteoglycan
‘ Anti-PG
information in regard to its various saccharide units (Table
I).
Native HSase TFMS
Origin -
200-
116-
66-
FIG.8. Immunochemical identification of native and deglycosylated GBM proteoglycan following polyacrylamide gel electrophoresis in SDS. Immunoblotting with antiserum against native proteoglycan (Anti-PG) was carried out as described under “Experimental Procedures.” The samples examined include the native as well as theheparitinase (HSase) and TFMS-treatedproteoglycan. The molecular weight markers are described in Fig. 1. TABLEI Molecular weight of bovine GBM heparan sulfate proteoglycan after deglycosylntwn by various chemical and enzymatic Drocedures Specificity of procedure” Treatment
None Nitrous acidd Heparitinased Heparitinase plus peptide N-glycosidase F TFMS’
Molecular weight‘
Heparan N-linked 0-linked unitsb units sulfate
-
+ + + +
-
+ +
6897
-
200,000 168,000 143,000 135,000
+ 128,000 Specificity is indicated by a plus sign to designate complete or partial removal of a particular typeof carbohydrate unit anda minus sign to indicate lack of effect on this unit. * Refers to GalNAc-Ser(Thr)-linked saccharide units. The apparentmolecular weights were calculated from electrophoretic migration of native and modified proteoglycan relative to standard proteins and must be considered as approximations. Nitrous acid removes only the peripheral portion of the heparan sulfate chains, whereas heparitinase releases all but a small internal segment. e The Asn-linked GlcNAc is conserved by this procedure, and only a partial removal of Ser(Thr)-linked GalNAc residues is achieved under the conditions employed (5). TFMS-prepared core protein retained 80% of the original activity. Immunoblotting with this anti-proteoglycan serum delineated bands in the deglycosylated preparations and in the intact proteoglycan (Fig. 8) which migrated to the same positions as those revealed by chemical radiolabeling of the peptide portion (Fig. 1). DISCUSSION
By employing enzymatic and chemical deglycosylation procedures of different specificities, we have been able in the present investigation to identify the core protein of the bovine GBM heparan sulfate proteoglycan and to provide further
Treatment of the proteoglycan with TFMS, a reagent which is known to degrade all carbohydrate units (5),yielded a single discrete polypeptide band with an apparent M , of128,000 which can be considered to represent the core protein. Indeed, the size of this product corresponds well with that expected from compositional analyses which have shown that saccharides constitute 30% of the proteoglycan’s weight. Furthermore, the loss of lectin-reactivity of the TFMS-treated molecule wasconsistent with effective deglycosylation. After heparitinase digestion the M , of the proteoglycan was decreased to an extent consistent with the removal of the four ( M , = 14,000) heparan sulfate chains which have been found in each molecule (4). A smaller reduction in size wasapparent after nitrousacid treatment (Table I), and thisdifference can be explained by the fact that the enzyme cleaves the glycosaminoglycan chains closer to the carbohydrate-protein linkage region than the chemical reagent. Indeed, the resistance of the internal segment of the heparan sulfate chains to nitrous acid degradation has been previously observed and attributed to an uneven distribution of N-sulfate groups (4, 24). The decrease in M , of about 8000 brought about by the peptide N-glycosidase F digestion is consistent with the removal of N-linked carbohydrate units from the proteoglycan (16). These oligosaccharides are believed to be exclusively of the complex type since no release was achieved by endo H treatment, and, indeed, DEAE-cellulose chromatography indicated that they were primarily acidic species. Since there are 8-9 mol of mannose/mol of proteoglycan (4), the presence of three complex asparagine-linked saccharide units/molecule is indicated by these data. The further decline in molecular weight brought about by TFMS treatment(about 7000) most likely represents a release of 0-linked saccharides, including the remaining stubs from the heparan sulfate chains (Table I); the occurrence of a number of small GalNAc-Ser(Thr)linked carbohydrate units on this GBMproteoglycan has previously been noted (4). It is apparent from the present study that since the GBM proteoglycan, like those from other sources (25,26), contains saccharide units other than the glycosaminoglycan chains, the removal of only the latterdoes not necessarily lead to the formation of a product that represents the polypeptide core. Indeed, a difference of 15,000 in molecular weight was observed between the heparitinase and TFMS-treated proteoglycan (Table I), and the loss of lectin interaction occurred only after the chemical deglycosylation. Presumably, the concanavalin A combines with the N-linked units of the proteoglycan,whereas a-D-galaCtOSyl residues on N- and/or 0attached saccharides are the binding sites for the B. simplicifolia I (27). The presence of the B. simplicifoliu I-reactive saccharides on the GBM proteoglycanis of particular interest since it represents yet another GBM constituent besides laminin (22) and Type IV collagen (2) which has been found to containa-D-galaCtOSylgroups and would therefore contribute to binding capacity of basement membranes for this lectin, which wasoriginally reported by Peters and Goldstein (28). The preparation of heparan sulfate-depleted and TFMSdeglycosylated proteoglycan providedus with an opportunity to assess the contribution of the saccharide units to the antigenicity of the molecule. Our data indicate that thepolyclonal response to the native proteoglycan is primarily directed against determinants on the polypeptide portion of the glycoconjugate, a finding that is in agreement with observa-
6898
Deglycosylation of Glomerular Basement Membrane Proteoglycan
tions on heparan sulfate proteoglycans from the EngelbrethHolm-Swarm sarcoma (29). The molecular weight of the core protein of the bovine GBM heparan sulfate proteoglycan clearly differs from that of the component which has been isolated from rat glomeruli (30), since after digestion of the latter with heparitinase a reduction in M , from 130,000 to 18,000 has been reported. The basement membrane-producing mouse EngelbrethHolm-Swarm sarcoma has yielded a low density (Mr > 500,000) anda high density (Mr = 350,000) proteoglycan which uponenzymatic removal of heparan sulfate chainshave been shown to yield molecules with M , values of 350,000400,000 and 95,000-130,000, respectively (7); in another study a molecular weight of about 10,000 has been reported for TFMS-prepared protein core of the high density glycoconjugate from the mouse tumor (29). Since the synthesis of glycosaminoglycans is a post-translational event (31), the difference in heparan sulfate chain lengths noted in proteoglycans from various sources (4, 7, 30, 32-34) is not surprising and can be attributed to variationin enzymatic machinery and substrateavailability. Although the diversity in core protein size observed in basement membrane proteoglycans might suggest a direct genetic basis, this may not necessarily be the case; as indicated by studies with Engelbreth-Holm-Swarm tumor cells, proteolytic processing of the heparan sulfate proteoglycan can occur (35), andsuch a mechanism may beresponsible for the formation of different core proteins from a common precursor, depending on species or cellular origin. Although the core proteins identified by deglycosylation procedures of mature proteoglycans are probably not the primary gene products, they do represent the physiologically relevant polypeptides which dictate the disposition of the carbohydrate units of the proteoglycans and influence their interaction with other basement membrane macromolecules. REFERENCES 1. Heathcote, J. G., and Grant, M. E. (1981) Znt. Rev. Connect. Tissue Res. 9,191-264 2. Mohan, P. S., and Spiro, R. G. (1986) J. Biol. Chem. 261,43284336 3. Farquhar, M. G. (1981) in Cell Biology of Extracellular Matrix (Hay, E. D., ed) pp. 335-378, Plenum Press, New York 4. Parthasarathy, N., and Spiro, R. G. (1984) J. Bwl. Chem. 2 5 9 , 12749-12755 5. Edge, A. S. B., Faltynek, C.R., Hof, L., Reichert, L.E., and Weber, P. (1981) Anal. Biochem. 118, 131-137
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