the of vol. 267, no. of 15, 5700-5711,1992 1992 printed in ... · 1 and lamp-2 antibodies were...

THE JOURNAL 0 1992 by The American Society for Biochemistry

OF BIOLOGICAL CHEMISTRY and Molecular Biology, Inc.

Vol. 267, No. 8, Issue of March 15, pp. 5700-5711,1992 Printed in U. S.A.

Differential Glycosylation and Cell Surface Expression of Lysosomal Membrane Glycoproteins in Sublines of a Human Colon Cancer Exhibiting Distinct Metastatic Potentials*

(Received for publication, August 12,1991)

Osamu SaitohS, Wei-Chun Wanglj, Reuben Lotad, and Minoru FukudaSII From the $La Jolla Cancer Research Foundation, Cancer Research Center, La Jolla, California 92037, the §Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, Washinzton 98121, and the llUniuersity of Teras, M. D. Anderson Cancer Center,

I

Houston, Teras 77030

Changes in the glycosylation of asparagine-linked oligosaccharides have been shown in various tumor cells, including human colon cancer. Attempts were made to elucidate the difference in Asn-linked oligosaccharides attached to lysosomal membrane glycoproteins isolated from sublines of human colon carcinoma exhibiting high and low metastatic potentials in nude mice. Lysosomal membrane glycoproteins (lamp) 1 and 2 were immunoprecipitated from the cells after labeling with radioactive sugars, and the glycopeptides prepared were fractionated by serial lectin affinity chromatography employing immobilized concanavalin A, Datura stramonium agglutinin, and tomato lectin. Comparison of Asn-linked oligosaccharides from the different colonic carcinoma cells revealed the following features. First, the highly metastatic carcinoma cells express more poly-N-acetyllactosaminyl side chains with branched galactose residues than cells with low metastatic potential. Second, sialylation is more significant in the highly metastatic carcinoma cells than in the poorly metastatic ones. Conversely, N- acetyllactosamine units are less fucosylated in the highly metastatic cells than in poorly metastatic cells. These structural changes were apparently caused by the increase in sialyltransferase and the decrease in al+3 fucosyltransferase in the highly metastatic cells. The results also suggest that highly metastatic carcinoma cells express more sialyl LeX structures at the termini of poly-N-acetyllactosaminyl side chains than poorly metastatic carcinoma cells. Further, highly metastatic cells were found to express more lamp-1 and lamp-2 on the cell surface. These results were found to be correlated to the increased expression of sialyl LeX structures with high affinity binding of anti-sialyl LeX antibody on highly metastatic cells. Increased expression of sialyl LeX in the poly-N-acetyllactosamines of the cell surface may contribute to the metastatic be- havior of the cells, assuming that this structure can serve as a better ligand for selectins present on endothelial cells and platelets.

Polylactosaminoglycans are high molecular weight carbo-

* This work was supported by Grant R01 CA48737 awarded by the National Cancer Institute. 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 solely to indicate this fact.

I( To whom correspondence should be addressed La Jolla Cancer Research Foundation, 10901 N. Torrey Pines Rd., La Jolla, CA 92037.

hydrates and are distinct from usual complex-type Asn-linked saccharides by having side chains composed of Gal@l+ 4GlcNAc@1+3 repeats, which are susceptible to endo-& galactosidase. Polylactosaminoglycans can carry various an- tigenic determinants such as I/i and AB0 blood groups (for review, see Refs. 1-3). Furthermore, the structures of polylactosaminoglycans are often characteristic to different cell types and stage of differentiation. For example, granulocytes and monocytes are enriched in the structures of Galj3l+ 4(Fucal4)GlcNAc/31+R, LeX, or NeuNAca2+3Gal@l+ 4(Fuca1+3)GlcNAc(3l+R, sialyl LeX, which reside at the termini of polylactosaminoglycans in these cells (4, 5) . Very recently, it was discovered in several laboratories that these unique structures of poly-N-acetyllactosamines in granulocytes and monocytes serve as ligands for adhesive molecules, selectins, in endothelial cells and platelets (6-9). Reports from several other laboratories demonstrated that the level of sialyl LeX or sialyl Le” NeuNAca2+3Gal~1+3(Fucal+4)Glc- NAc+R are increased in tumor cells, including carcinomas (10-12).

It has been repeatedly observed that malignant transformation of rodent and human cells is associated with an increase in the amount of tetraantennary and triantennary N-glycans containing side chains, which are linked to a- mannose through a GlcNAcP14 linkage (13-15). In particular, the transformation of rat2 fibroblasts with oncogenes such as T24H-ras, V-K-ras and V-fps results in the increased N-acetylglucosaminyltransferase V, which forms the above antenna and acquisition of invasive and metastatic potential (16, 17). This increased amount of the tetraantennary and triantennary N-glycans is often associated with the increased amount of poly-N-acetyllactosamine. This is because p1-3- N-acetylglucosaminyltransferase, the key enzyme for the formation of N-acetyllactosaminyl repeats, preferentially acts on the side chains branched from mannose via GlcNAc/31+ 6, which is formed by N-acetylglucosaminyltransferase V (18).

We have shown previously that lysosomal membrane glycoproteins, lamp-1’ and lamp-2, are the major carriers of polylactosaminoglycans in various cells (19, 20). It was also demonstrated that lamp-1 is the major glycoprotein containing GlcNAcf i l4Manal4Man branching in metastatic tumor cells as detected by leukophytohemagglutinin binding (21,22). It is becoming evident that some of lamp-1 and lamp- 2 are expressed on the cell surface, although the majority of these molecules reside in lysosomes (20,23-25). These results

The abbreviations used are: lamp-1 and lamp-2, lysosomal membrane glycoproteins 1 and 2; ConA, concanavalin A; DSA, Datura stramonium agglutinin; PBS, phosphate-buffer saline; BSA, bovine serum albumin; Fuc, fucose.

5700

Lysosomal Membrane Glycoprotel

prompted us to examine the carbohydrate structures of lamp- 1 and lamp-2 isolated from highly metastatic and poorly metastatic sublines derived from a human colon tumor, in order to determine whether lamp-1 and lamp-2 are the major carriers for polylactosaminoglycans on the cell surface of these tumor cells, and whether the glycosylation of these glycoproteins is different in cells exhibiting distinct metastatic potentials.

EXPERIMENTAL PROCEDURES

Cell Lines-Four cell lines derived from a single human colon carcinoma specimen, designated KM12-C, KM12-SP, KM12-SM, and KM12-L4, were kindly provided by Dr. Isaiah J. Fidler (M. D. Anderson Cancer Center, Houston, TX). After intrasplenic injection, KM12-C and KM12-SP are poorly metastatic to liver, whereas KM12-SM and KM12-L4 are highly metastatic to liver (26, 27). The sublines were established from a primary colon carcinoma (Duke's stage B2) as described previously (26) and shown schematically in Fig. 1. Briefly, tumor tissue was dissociated into single cells or small cell clumps by treatment with collagenase type I and deoxyribonucle- ase. The cell suspension was divided into several aliquots, one of which was used directly to establish the cell line designated KM12- C. Other aliquota of the same cell suspension were injected into the spleen of several male athymic BALB/c nude mice, and cell line KM12-L4 was developed after four cycles of intrasplenic injections and production of experimental hepatic metastases in nude mice (see Fig. 1). Cell line KM12-SM was derived from a rare liver metastasis produced by parental KM12-C cells growing in the cecum wall of a nude mouse (Fig. 1) (27).

Cell Culture and Metabolic Labeling-The carcinoma cell lines were cultured in RPMI 1640 medium containing 10% fetal calf serum, 1% sodium pyruvate (11.0 mg/ml), 1% penicillin (10,000 units/ml), and streptomycin (10 mg/ml). For metabolic labeling, the cells were cultured in 98% of RPMI 1640 glucose-deficient medium supple- mented with 10% dialyzed fetal calf serum and 2% of regular RPMI 1640 that contained 10% fetal calf serum (final glucose concentration, 0.28 mM) (28). [3H]Glucosamine and [3H]galactose, or [3H]fucose or [3H]mannose were added at a concentration of 10 pCi/ml and 20 pCi/ ml, respectively, and cells were metabolically labeled for 24 h a t 37 "C.

Immunoprecipitation and Sodium Dodecyl Sulfate-Polyacrykamide Gel Electrophoresis-Radiolabeled cells were lysed in PBS (6.7 mM potassium phosphate buffer, pH 7.4, 150 mM NaCl, 0.002% NaN3) containing 1% Nonidet P-40 and protease inhibitors as described (28). Lamp-1 and lamp-2 were sequentially immunoprecipitated from the cell lysates by addition of rabbit anti-human lamp-1 antibodies or rabbit anti-human lamp-2 antibodies as described (28). Anti-lamp- 1 and lamp-2 antibodies were kindly provided by Dr. Sven Carlsson, University of Umei, Umei, Sweden. Aliquots of radiolabeled samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a 7.5% polyacrylamide gel according to Laemmli (29),

1

t t

uver-

culhrre 1

+ 4 CUilUIE culture

t Culture

i hlghlymetartatic E l *

FIG. 1. Experimental procedures used for the selection and isolation of variant cell lines with different metastatic potential. Figure was adapted from Figs. 1 and 2 in Ref. 26.

ins in Metastatic Carcinoma Cells 5701

followed by fluorography using Enlighting (Du Pont-New England Nuclear).

Lectin Affinity Chromatography of Glycopeptides-Glycopeptides were released by Pronase digestion of immunoprecipitates and fractionated by serial lectin affinity chromatography employing concanavalin A (ConA)-Sepharose, tomato lectin-Sepharose, and Datura stramonium (DSA)-agarose. The conditions for these chromatogra- phies were described previously (28, 30). Briefly, the glycopeptides were first applied to a column of ConA-Sepharose, and complex-type triantennary and tetraantennary asparagine-linked glycopeptides (Fraction I) were separated from biantennary asparagine-linked glycopeptides (Fraction 11) and high-mannose glycopeptides (Fraction 111). Fraction I that contains tri- and tetraantennary glycopeptides was then applied to a column of tomato lectin-Sepharose. The glycopeptides that bound to this column, IB, contain poly-N-acetyllactosaminyl side chains with three or more N-acetyllactosamine repeats (28,30). Glycopeptides unbound to tomato lectin-Sepharose, IA, were then applied to a column of DSA-agarose. DSA-agarose binds glycopeptides containing N-acetyllactosamine repeats and/or containing R4lcNAc@1+6(R4lcNAc~l+2)Man- branchings (Fraction IA2) (31).

Endo-@-galactosidase Treatment-Radiolabeled glycopeptides were digested with endo-@-galactosidase under the conditions described

freundii (32) and kindly donated by Dr. Michiko N. Fukuda of our previously (28). Endo-@-galactosidase was purified from Escherichia

institute. In the second set of experiments, radiolabeled glycopeptides were hydrolyzed in 0.1 N trichloroacetic acid at 100 "C for 1 h (4) before they were subjected to the treatment by endo-@galactosidase. The hydrolysate was neutralized by 1 N NaOH and desalted by Sephadex G-15 gel filtration using water as eluent. This acid hydrolysis removes both fucose and sialic acid.

Methylation Analy~k-[~H]Mannose-, [3H]galactose-, or [3H]glucosamine-labeled glycopeptides were methylated, together with un- labeled fetuin glycopeptides as carriers by the procedure of Hakomori (33) as described previously (28). The methylated galactose, mannose, and fucose residues were separated directly by thin layer chromatography as described (28). The methylated glucosamine derivatives were N-acetylated (4) and then separated by thin layer chromatography on a Silica Gel G, using the solvent system of acetone/water/ammo- nium hydroxide (2503:1.5, v/v/v). The sample lane was separated into 0.5-cm sections, and the radioactivity was determined by scintil- lation counting. Methylated standards of [3H]glucosamine were obtained from lamp-1 IB glycopeptides of HL-60 cells (28).

Estimation of the Number of Sialic Acid Residues-In order to estimate the number of sialic acid residues/mole of glycopeptide, glycopeptides were applied to a column of QAE-Sephadex A-25 equil- ibrated with 10 mM pyridine/acetic acid buffer, pH 5.5. After washing with the same buffer, monosialo, disialo, trisialo, tetrasialo, and pentasialo (plus more highly sialylated) glycopeptides were eluted step-wise in 20, 70, 150, 200 mM NaCl, and 1 M NaCl in the same buffer (28).

Assay of Glycosyltransferases-All enzymes except a 1 4 fucosyltransferase were assayed under the same conditions as described previously (28, 34). Briefly, Gal@1-*4GlcNAc@1+3Gal@l+ 4GlcNAc@1+3Ga1@14G1c~14-(CH2),CH3 and GlcNAc@l+ 2Mana14Gl~@l+O-(CH~)~CH~ (35) were used as acceptors for UDP-GlcNAc:@-Gal @1+3-N-acetylglucosaminyltransferas.e, "extension enzyme," and UDP-G1cNAc:a-Man @14-N-acetylglucosaminyltransferase (V), respectively. These two substrates were kindly provided by Dr. 014 Hindsgaul, University of Alberta, Canada. Asialo a,-acid glycoprotein was used as an acceptor for CMP-NeuNAc @- Gala2-3 sialyltransferase and a 2 4 sialyltransferase and the product was treated by Newcastle disease virus neuraminidase as described (34). The radioactivity remaining after neuraminidase digestion was regarded as the product due to a 2 4 sialyltransferase, whereas the difference between nontreated and neuraminidase-treated producta was regarded as the product due to a2+3 sialyltransferase (34). In order to measure the activity of al+3 fucosyltransferase, Fucal- 2Gala14Glc was used as an acceptor under the conditions described (36).

Estimation of Number of Lamp-1 and Lamp-2 Molecules and Sialyl L? Structures on the Cell Surface-To estimate the number of lamp- 1 and lamp-2 molecules and sialyl LeX termini on the surface of the colon tumor cell lines, the number of binding sites of monoclonal antibodies was measured according to the procedure of Ho and Springer (37). Since monoclonal antibody specific to sialyl LeX (CSLEX1, provided by UCLA tissue typing laboratory, Ref. 10) is an IgM, the sandwich method was used for the determination of binding

5702 Lysosomal Membrane Glycoproteins in Metastatic Carcinoma Cells

sites. Thus, adherent carcinoma cells were washed with PBS containing 4 mg/ml BSA, and then various amounts of the monoclonal antibodies were added. After incubation at 4 “C for 1 h, the cells were washed with cold PBS containing BSA and ‘*’I-labeled goat anti- mouse IgM diluted in PBS containing BSA was added. In preliminary experiments, the amount of the second antibody was determined to saturate the binding sites. The number of binding sites was calculated from the determination of the specific activity by measuring the total radioactivity of the aliquots used for each experiment. Similarly, the binding sites of lamp-1 and lamp-2 were estimated by using mouse monoclonal antibodies BB6 (specific to lamp-1) and CD3 (specific to lamp-2, Ref. 20) followed by the addition of ‘251-labeled goat anti- mouse IgG. ‘*’I-Labeled goat anti-mouse IgM and anti-mouse IgG were purchased from Du Pont-New England Nuclear. In order to estimate the binding sites a t the cell surface, the whole procedure was carried out on ice. In order to estimate the binding sites including the cytoplasm, the cells were fixed with 0.5% formaldehyde, and permeabilized with 0.5% saponin, as described (38). The monoclonal antibodies were then added on the fixed, permeabilized cells on ice.

Indirect Immunofluorescence-Indirect immunofluorescence was carried out exactly as described previously (38). Briefly, the cells were fixed first with 0.5% formaldehyde in PBS for 20 min at room temperature and then permeabilized with 0.5% saponin and 4 mg/ml BSA in PBS. The fixed and permeabilized cells were then incubated with anti-lamp-1 or lamp-2 monoclonal antibody followed by fluorescein isothiocyanate-conjugated F(ab’), fragment of goat anti-mouse IgG. Alternatively, the cells were incubated on ice with appropriate antibody, then fixed with paraformaldehyde. The cells were then incubated in the second antibody conjugated with fluorescein isothiocyanate: The latter procedure is to detect lamp molecules on the cell surface while the former procedure can detect the intracellular lamp molecules as well. Similarly, the cells were incubated with mouse anti-sialyl Le” monoclonal antibody (purchased from Signet Labora- tories, Dedham, MA) followed by fluorescein isocyanate-conjugated F(ab’), fragment of goat anti-mouse IgG.

RESULTS~

Lamp-1 and Lamp-2 from High and Low Metastatic Colonic Carcinoma Cells-The colonic carcinoma cell lines with vary- ing metastasic potentials were metabolically labeled in the presence of one of the radioactive monosaccharides, and lamp- 1 and lamp-2 were then immunoprecipitated. As shown in Fig. 2A, the major glycoproteins in the total cell lysates are glycoproteins with M , -170 to -180 and M , -130. The latter glycoproteins correspond to lamp-1 and lamp-2 as shown in Fig. 2B, indicating that lamp-1 and lamp-2 are the major glycoproteins even in the total cell lysate. Fig. 2 also reveals that [‘H]fucose was incorporated more in the poorly metastatic cells than in the highly metastatic cells (compare SP versus LA in Fig. 2). Examination by sodium dodecyl sulfate- gel electrophoresis did not reveal an obvious difference in the molecular weight of lamps between highly metastatic and poorly metastatic colonic carcinoma cells.

Fractionation of Glycopeptides by Serial Lectin Affinity Chromatography-The difference in carbohydrate moieties attached to lamps was demonstrated when the glycopeptides were fractionated by serial lectin chromatography. Glycopep- tides were prepared from immunoprecipitated lamp-1 and lamp-2, and the radioactive glycopeptides were fractionated as shown in Fig. 3. The results indicate clearly that lamp-1 from the highly metastatic colon carcinoma cells, L4, yield a greater proportion of glycopeptides that are bound by tomato lectin-Sepharose than glycopeptides from the poorly metastatic SP cells. Similar results were obtained when lamp-2 glycopeptides were analyzed as shown in Fig. 4. Furthermore, the highly metastatic cells (LA) contained a higher amount of

Portions of this paper (including part of “Results,” Figs. 4-12, and Tables I-V) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

A GlcN Fuc ”

116- 97-

66 -

45-

B Lamp-1 Lamp2 ”

- 2w

’ -116 - 97

-86

- 45

FIG. 2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total cell lysates ( A ) and lamp-1 and lamp-2 ( B ) after labeling with various radioactive sugars. After labeling, the total cell lysates ( A ) or lamp-1 and lamp-2, sequentially immunoprecipitated from the total lysates (B), were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by fluorography. In experiment A, 100 pg of protein was applied to each lane. The arrowhead indicates the migration positions of lamp-1 and lamp-2. In experiment B, [3H]mannose-labeled samples are shown. Similar results were obtained on [3H]glucosamine, [‘H]galactose, or [3H]fucose-labeled samples. The migrations of standard proteins are indicated at the margins.

0 1 0 io -0 1 0 2 0

FRACTION

FIG. 3. Lectin affinity chromatography of [3H]glucosamine- labeled glycopeptides from lamp-1 synthesized by poorly metastatic (SP) and highly metastatic (L4) colon carcinoma cells. The samples of lamp-1 shown in Fig. 2B were digested by Pronase, and the radiolabeled glycopeptides were sequentially applied to ConA- Sepharose, tomato lectin-Sepharose, and DSA-agarose, as described under “Experimental Procedures.” Haptene sugars were added at the points indicated by arrows within graphs. Aliquots were taken for determination of radioactivity. The samples were pooled as indicated by horizontal bars.

Lysosomal Membrane Glycoproteins in Metastatic Carcinoma Cells 5703

glycopeptides, which bound to DSA-agarose (Fraction fA2), in both lamp-1 and lamp-2 than the poorly metastatic SP cells (Figs. 3 and 4 and Table I). Another pair of highly and poorly metastatic cell lines, SM and C, was also metabolically labeled with [3H]galactose and [3H]mannose, and glycopeptides were prepared from immunoprecipitated lamp-1 and lamp-2, As shown in Table I, the highly metastatic cell line ( S M ) again yielded a greater proportion of glycopeptides bound to tomato lectin-Sepharose and DSA-agarose than the poorly metastatic cell line (C). These results indicate that highly metastatic colon carcinoma cells contain more glycopeptides bound to tomato lectin-Sepharose and to DSA-Seph- arose than poorly metastatic colon carcinoma cells.

Structures of Asparagine-linked Oligosaccharides-To un- derstand the difference in Asn-linked oligosaccharides between high and low metastatic colon carcinoma cells, the structural analysis of glycopeptides was carried out as shown in the Miniprint. To simplify the description of the structural analysis, the glycopeptides obtained from lamps of the highly metastatic carcinoma cell line L4 are described first in detail.

Structures of IB Glycopeptides-Based on the results described in the Miniprint, the structures of the glycopeptides bound to the tomato lectin-Sepharose column can be proposed, as shown in Fig. 13A. IB glycopeptides are tetraantennary saccharides which contain 5 to 6 mol of N-acetyllactosaminyl units/mol of glycopeptides, thus 1-2 mol of N - acetyllactosamine are present in poly-N-acetyllactosaminyl extension. The longest poly-N-acetyllactosaminyl extension has the backbone of linear, Gal@14GlcNAc@l+3Gal@l+ 4GlcNAc@l+3Gal@1+4G1cNAc@l+Man or branched structure, Gal@l4GlcNAc@l+3(Gal@l4GlcNAc@l+6)Gal@1+ 4GlcNAc@l+Man. 63% of the N-acetyllactosaminyl units are substituted with cu1-3 fucose residues, which are apparently attached uniformly to N-acetylglucosamine residues at various positions. In addition, 0.4 mol of the terminal galactose is substituted with al+2-linked fucose. The majority of IB glycopeptides contain 2 or 3 sialic acid residues. Together, the termini of IB glycopeptides contain sialyl LeX structure, NeuNAc~2+3Gal@l+4(Fucal+3)GlcNAc and LeY structure, Fucal+2Gal@l+4(Fuca1+3)GlcNAc. Portions of these structures were released by endo-@-galactosidase (Fig. 7).

In Fig. 13, the structures were proposed for the glycopeptides that contain Gal@1+4GlcNAc, namely type 2 chain in the side chains. However, it has been reported that some of the oligosaccharide side chains in colonic epithelial cells could contain the Gal@l+3GlcNAc structure, namely the type 1 chain (39). In order to estimate the ratio of Gal@ldGlcNAc to Gal@l+3GlcNAc linkages, the desialylated, defucosylated IB glycopeptides were digested by @-galactosidase and @-N- acetylglucosaminidase, both purified from Diplococcus pneu- monia. Diplococcal @-galactosidase was shown to hydrolyze only Gal(314GlcNAc linkage but not Gal@l+3GlcNAc linkage (40). The results indicate that more than 70% of the side chains are composed of Gal@l4GlcNAc linkages (data not shown). This number needs to be taken as a minimal estimate since diplococcal 8-N-acetylglucosaminidase may not cleave all of the GlcNAc@l+4Gal@l4G1cNAc~l+4/6Man~R structures (39). In order to determine whether these cells express type 1 structures, we examined the cells by indirect immunofluorescence using anti-sialyl Le" antibody. The results showed that both SP and L4 cells express a small but detectable amount of sialyl Le" structure (data not shown). These results therefore indicate that the great majority of the side chains consists of N-acetyllactosamine units (type 2 chain), whereas a small portion of the side chains (less than 30%) have Gal@l+3GlcNAc structures (type 1 chain).

~ P I - ~ G I E N A E ~ I ' T

fFuCrzl

FIG. 13. Proposed structures of Asn-linked glycopeptides obtained from lamp-1 and lamp-2 in highly and poorly metastatic carcinoma cells. Proposed structures for major saccharides in IB glycopeptides ( A ) , IA2 glycopeptides (B), and IA1 glycopeptides ( C ) are shown. On the average, IB glycopeptides contain 5-6 N- acetyllactosaminyl units with a branched galactose. IA2 and IA1 glycopeptides contain, on the average, 4-5 N-acetyllactosamine units. About 20% of terminal galactose residues is substituted with (~1-2 fucose instead of sialic acid residues. A very small portion of N- acetyllactosaminyl side chains are composed of type 1 chain, Galj31+ 3GlcNAc. IA1 glycopeptides are extensively fucosylated at N-acetyllactosamine units. The highly metastatic cells ( L 4 ) are more sialylated and less fucosylated than the poorly metastatic cells ( S P ) (see text for details).

Structures of IA2 Glycopeptides-Based on the results described in the Miniprint, the structures of IA2, the glycopeptides that were bound to DSA-agarose, are proposed as shown in Fig. 13B. IA2 glycopeptides have on the average 4.6-4.9 mol of N-acetyllactosaminyl units in tetraantennary structures, thus 0.6-0.9 mol of N-acetyllactosaminyl units are present as poly-N-acetyllactosaminyl extensions. The majority of IA2 glycopeptides contains 4,3, or 2 sialic acid residues. IA2 glycopeptides contain the least amount of fucose residues attached to N-acetyllactosaminyl side chains among IB, IA1, and IA2 glycopeptides. A high proportion of N-acetyllactosamines directly attached to a-mannose are non-fucosylated N - acetylglucosamines. Otherwise, endo-@-galactosidase treatment should have shown a greater release of oligosaccharides after defucosylation (see Fig. 10). Fucose residues therefore must be attached preferentially to the external positions of N-acetyllactosaminyl side chains. In addition, fucose is attached to the terminal galactose. As a whole, IA2 glycopeptides therefore have sialyl LeX and LeY terminal structures.

Structures of I A l Glycopeptides-Based on the results described in the Miniprint, the structures of IA1 glycopeptides that were not bound to DSA-agarose are proposed as shown in Fig. 13C. The majority of IA1 glycopeptides are tetraantennary saccharides containing 0 or 1 mol of poly-N-acetyllac-


tosaminyl extensions. IA1 glycopeptides contain, on the average, 2 sialic acid residues. IA1 glycopeptides are highly fucosylated and more than 75% of N-acetyllactosaminyl units are substituted with fucose. These fucose residues are uniformly distributed. Thus, a majority of IA1 glycopeptides must have at least 1 mol of sialyl LeX and a maximum of 0.5 mol of LeY/mol of the glycopeptides.

Comparison of Glycopeptide Structures from Poorly Met- astatic and Highly Metastatic Colon Carcinoma Cell Lines- Glycopeptides obtained from poorly metastatic cells were subjected to structural analysis in the same way as was done for those from highly metastatic cells, and the following conclusions can be drawn. (a) Lamps from highly metastatic cells contain more polylactosamine-containing Asn-linked oligosaccharides, both IB and IA2 glycopeptides, than poorly metastatic carcinoma cells (Table I). Concomitantly, much less IA1 glycopeptides were obtained from highly metastatic cells compared to poorly metastatic cells. (b) The glycopeptides from highly metastatic carcinoma cells are appreciably more sialylated than the counterparts from poorly metastatic cells (Table IV). Conversely, the fucose content is much higher in the glycopeptides isolated from poorly metastatic carcinoma cells (Tables I11 and V). Methylation analysis and fucose content indicated that IA1 glycopeptides from poorly metastatic carcinoma cells contain almost fully fucosylated N-acetyllactosamine units. (c) Since ConA I and ConA I1 glycopeptides contain 3 mol of mannose/mol of glycopeptides, the number of Asn-linked oligosaccharides can be estimated as shown in Table VI, assuming that high mannose oligosaccharides contain about 7.5 mol of mannose/molecules as shown in the previous studies for HL-60 cells (28). The results indicate that the major difference between highly and poorly metastatic cells is that more sialylated but less fucosylated in the N-acetyllactosamines in the highly metastatic carcinoma cells. In addition, slightly more N-glycosylation sites are modified to acquire poly-N-acetyllactosamine in the highly metastatic cells. On the other hand, the number of N-acetyllactosamine units in each glycopeptide fraction does not differ irrespective of the metastatic potential of the cells.

Comparison of Glycosyltransferase Activities between Highly and Poorly Metastatic Colon Carcinoma Cell Lines-To un- derstand further the mechanisms underlying the difference in glycosylation, ~-Gal-~l+3-N-acetylglucosaminyltransferase,

TABLE VI Asn-linked oligosaccharide chains attached to lamp-1 and lamp-2

from low metastatic (SP) and high metastatic (L4) colonic tumor cell lines

The numbers are expressed as moles/lamp-1 or lamp-2 molecule, and were calculated based on the recovery of [3H]mannose-labeled glycopeptides (Table I).

Lamp-1 Lamp-2 Oligosaccharide (18)’ (16)”

SP LA SP L4

Complex-type containing polylactosa- 0.5 1.9 0.6 1.7 mine with 3 2 repeats of N-acetyllactosamine (IB)

Complex-type containing polylactosa- 1.3 6.0 1.6 6.4 mine with 1-2 repeat(s) of N-acetyllactosamine (IA2)

Heavily fucosylated complex-tyep tri- 14.1 7.9 12.2 6.2

Biantennary complex-type (11) 0.2 0.3 0.2 0.2 High mannose-type (111) 1.8 2.0 1.4 1.5

ecule (see the previous report of Ref. 20).

and tetraantennas (IA1)

The number of total carbohydrate chains/lamp-1 or lamp-2 mol-

extension enzyme, a-man-/314-N-acetyIglucosaminyltrans- ferase, “GlcNAc-transferase V,” P-Glc(NAc)-al+3 fucosyltransferase, a2+3, and a 2 4 sialyltransferases were assayed. Although no appreciable difference was observed in the first two enzymes, the latter two enzymes show a dramatic difference between high and low metastatic cells. Table VI1 reveals that poorly metastatic SP cells express twice as much (~1-3 fucosyltransferase as highly metastatic L4 cells, whereas highly metastatic L4 cells express 50% more a2+3 sialyltransferase than poorly metastatic SP cells. These results are in good agreement with the above results that the highly metastatic cells contain more sialylated but less fucosylated glycopeptides than the low metastatic cells.

Comparison of Cell Surface Lamp Expression between High and Low Metastatic Carcinoma Cells-It has been shown that a small portion of lamp molecules can be expressed on the cell surface, and these molecules can be the carriers of poly- N-acetyllactosamine chains on the cell surface (20, 23-25). To test if highly metastatic carcinoma cells express more lamp on the cell surface, the expression of lamp molecules was examined by indirect immunofluorescence. As shown in Fig. 14A, the intensity of immunofluorescence staining of the lamp-1 molecule on the surface of the poorly metastatic cells was relatively weak. In contrast, the immunofluorescence on the surface of highly metastatic cells was obvious and stronger than that on the poorly metastatic ones (compare C with A

TABLE VI1 Glycosyltransferase activities in extracts of poorly and highly

metastatic colonic cells Poorly Highly

SP cells L4 cells Enzyme metastatic metastatic

nmollh. mg protein UDP-G1cNAc:bGal j31+3-N-ace- 0.56 0.51

tylglucosaminyltransferase (extension enzyme)

tylglucosaminyltransferase (GlcNAc-transferase V)

yltransferase

transferase

transferase” a The amount of this enzymatic activity is almost close to the limit

UDP-G1cNAc:a-Man fi14-N-ace- 0.037 0.030

GDP-Fuc:/Y-Glc(NAc) ~~1+3-fuCOs- 0.42 0.20

CMP-NeuNAc:@-Gala2+3-sialyl- 0.12 0.19

CMP-NeuNAc:&Gal a2-6-sialyl- 0.01 0.01

of the detection.

FIG. 14. Expression of lamp-1 molecules on the cell surface and in the cytoplasm of poorly metastatic and highly metastatic colonic carcinoma cells examined by immunofluorescence. SP ( A and B ) and L4 (C and D) cells were examined either as intact cells for surface expression of lamp-1 ( A and C) or after permeabilizing with saponin for visualization of the intracellular distribution of lamp-1 ( B and D). The cells were incubated with monoclonal anti-(h-lamp-1) antibody followed by fluorescein isothiocyanate-conjugated goat anti-mouse IgG. The bar indicates 20 nm.


in Fig. 14). When the cells were permeabilized, intracellular lysosomal structures were stained with similar intensity in both cell types (Fig. 14, B and D). These results suggest that more lamp molecules are exposed on the surface of highly metastatic carcinoma cells than on poorly metastatic ones.

In order to quantitate this difference, the number of lamp- 1 and lamp-2 molecules was estimated by determination of the number of antibody-binding sites. As shown in Fig. 15A, more of the antibodies were bound to the surface of highly metastatic cells than poorly metastatic cells. The number of binding sites/cell was found to be 2.2 X lo4 and 1.4 X lo4 for highly and poorly metastatic cells, SP and L4, respectively (Table VIII). A similar difference was also found between another pair of cells, and 2.4 X lo4 and 1.2 X lo4 lamp-l/cell, respectively, were found to express on the cell surface of SM and C cells. In these experiments, nonspecific binding was less than 2% of the total binding. In addition, the size of the highly and poorly metastatic cells is very similar. We also estimated the number of lamp-1 and lamp-2 molecules in the whole cell, including the cytoplasm by the analysis of permeabilized cells. Table VI11 reveals that highly metastatic cells

o k 7 " - - J Mo eon

ng I well

FIG. 15. Saturation binding of antibodies recognizing lamp- 1, lamp-2, or sialyl Lex to low and high metastatic colonic carcinoma cells. Increasing concentration of monoclonal antibodies specific to lamp-1 ( A and C) or sialyl Lex ( B ) were incubated with cells in 96 wells, and binding was determined as described under "Experimental Procedures." In experiments A and B the sites on the cell surface were determined, while the sites in the whole cell including the cytoplasm were determined in C. In experiment B, the dotted lines were plotted to determine the binding sites with high affinity to the anti-sialyl Lex antibody. Open circle, poorly metastatic SP cells; closed circles, highly metastatic L4 cells.

TABLE VI11 Number of binding sites to monoclonal antibodies specific

to hmp-1, [amp-2, and sialyl Lex The numbers are expressed as 104/cell.

SP L4 C SM Lamp-1 Surface 1.40 2.17 1.21 2.42 (BB6) (4.5%)" (5.1%) (3.8%) (5.9%)

Total 31.14 42.70 32.07 40.82

Lamp-2 Surface 1.33 2.03 1.01 1.75 (CD3) (5.6%) (6.2%) (3.7%) (5.3%)

Total 23.68 32.74 26.97 32.80

Sialyl LeX Surface 4.20 4.15 3.20 3.95 (CSLEX1) (1.55)* (1.95)' (1.54)b (2.11Ib

*The numbers in parentheses indicate a proportion of lamp molecules on the cell surface expressed as a percentage of total lamp molecules. ' The numbers in parentheses indicate the binding sites with high

affinity to the monoclonal antibody.

contain slightly more total lamp-1 and lamp-2 molecules than poorly metastatic cells in both pairs of sublines (see also Fig. 15C).

Comparison of Cell Surface Sialyl Lex Expression between Highly and Poorly Metastatic Carcinoma Cells-The number of sialyl LeX determinants on the cell surface was estimated in order to test whether the expression of cell surface sialyl LeX correlates with the expression of lamp-1 and lamp-2 molecules. Table VI11 shows that the number of binding sites for anti-sialyl LeX antibodies/cell is close to that estimated for lamp-1 and lamp-2. Simple calculations, based on the number of binding sites, suggest that as much as 30% of sialyl LeX structures can be carried by cell surface lamp molecules. However, the anti-sialyl LeX antibody is of the IgM type, so that some antibodies could bind pentavalently, whereas some of the anti-lamp antibody binds divalently due to its IgG nature. Because of this difference in valency, these experiments do not provide the actual number of sialyl LeX determinants carried by cell surface lamp molecules.

Fig. 15B and Table VI11 also reveal that the highly metastatic cell expresses more of sialyl LeX structures with high affinity to the monoclonal antibody. This difference in the binding curve was reproduced in repeated experiments. This result is consistent with the above findings that highly metastatic cells express more of poly-N-acetyllactosaminyl glycopeptides (see Table VI), assuming that sialyl LeX structures at the termini of poly-N-acetyllactosaminyl side chains have higher affinity to the monoclonal antibody than those at short chains. The same experiments also showed that the number of sialyl LeX sites is almost the same at saturating antibody concentrations, regardless of cell types. This is probably due to the fact that low metastatic cells express more of shorter N-glycans enriched with sialyl LeX structure (IA1 glycopeptides). As a result, both highly and poorly metastatic tumor cells express a similar amount of sialyl LeX structure which has low affinity to the anti-sialyl LeX antibody.

DISCUSSION

The present studies revealed differences in Asn-linked oligosaccharides attached to lysosomal membrane glycoproteins 1 and 2 synthesized in poorly and highly metastatic human colon carcinoma cells. The results can be summarized as follows. First, the highly metastatic colon carcinoma cell lines (L4 and SM) synthesize more of Asn-linked oligosaccharides that contain poly-N-acetyllactosamines than their poorly metastatic counterparts (C and SP). Second, the po1y-N- acetyllactosamine chains are sialylated more extensively in


the highly metastatic colonic cells. Third, the poly-N-acetyllactosamine units are fucosylated to a lower degree in the highly metastatic colonic carcinoma cell lines. Table VI illus- trates that the number of Asn-linked oligosaccharides containing poly-N-acetyllactosaminyl units with branched galactose moieties (IB glycopeptides) increases from 0.5 to 1.9 mol for lamp-1 and from 0.6 to 1.7 mol for lamp-2, after the cells are selected for liver metastasis. The number of Asn-linked oligosaccharides containing linear N-acetyllactosaminyl repeats but fewer fucose residues (IA2 glycopeptides) increases dramatically after the same selection. Concomitant with this increase, the amount of oligosaccharides with a minimum amount of poly-N-acetyllactosamines but with higher fucosylation (IA1 glycopeptides) decreases dramatically in the highly metastatic cells (Table VI). These results indicate that the degree of sialylation increases but fucosylation decreases in the highly metastatic cell lines. These results were obtained because the highly metastatic cells express an increased amount of a2-3 sialyltransferase and a decreased amount of a1-3 fucosyltransferase than the poorly metastaic cells (Table VII). In addition, the increased sialylation may result in the decreased fucosylation in the highly metastatic colonic carcinoma cells, since sialylation in general hinders fucosylation at N-acetyllactosamine units and fucosylation follows after sialylation (42, 43). On the other hand, it is not clear at this point how the highly metastatic cells express a slightly increased amount of poly-N-acetyllactosamine. Consistent with our results it has been reported that a B16 melanoma variant, which is resistant to wheat germ agglutinin, is enriched in the Galp14(Fucal+3)GlcNAc structure but contains only a trace amount of sialic acid (44).

Our studies demonstrated that serial lectin affinity chromatography is useful in fractionating glycopeptides exhibiting differences in poly-N-acetyllactosamine. As reported previously (28,30), tomato lectin-bound fraction was preferentially enriched with glycopeptides containing longer poly-N-acetyllactosaminyl repeats. All of the glycopeptides with branched galactose were apparently bound to this immobilized lectin. In contrast, DSA-agarose can bind oligosaccharides even with short poly-N-acetyllactosamines. However, it is significant to note that the glycopeptides with short poly-N-acetyllactosamines were not bound to DSA when they were also heavily fucosylated, which is consistent with a previous report (31). It was reported that triantennary oligosaccharides lacking 2,6-di-O-substituted a-mannose also fails to bind to DSA (45). The present studies extend these observations in that they show not only the extent of poly-N-acetyllactosamine and the number of antennae but also other substitutions, such as fucosylation, can be discriminated efficiently by the serial lectin affinity chromatography employing ConA, tomato lectin, and DSA. It is worthwhile to mention that the effect of fucose substitution was not evident in fractionation of glycopeptides from HL-60 lamp molecule (28). This is most likely because glycopeptides from HL-60 lamps contain longer poly- N-acetyllactosaminyl side chains with less fucose substitution than those from colon carcinoma cells.

The present results are consistent with the previous reports on the expression of polylactosaminoglycans in transformed cells. It has been shown that baby hamster kidney cells transformed by the Rous sarcoma virus or polyoma ViNS express more polylactosaminoglycans than their untrans- formed parent cell lines (13-15). It is noteworthy that metastatic tumor cells transformed by various oncogenes also increase in the synthesis of the side chain arising from GlcNAc/314Manal-&, which is formed by N-acetylglucosaminyltransferase V (16,17). Since this side chain is the most

preferable site for poly-N-acetyllactosamine extension by ~1+3-N-acetylglucosaminyltransferase, the increased amount of the GlcNAc@l4Mand+R branch leads to an increase in poly-N-acetyllactosamine (18,45,46). Conversely, it was reported that tumor metastasis in animal models was reduced by the addition of glycosylation inhibitors such as swainsonine and castanospermine (47-49). Since these two inhibitors block the formation of side chains elongating from C-6 of a-mannose (50), they presumably inhibit poly-N-acetyllactosamine formation. Thus, the inhibition of poly-N- acetyllactosamine synthesis apparently reduces tumorigenicity. Similarly, we have shown recently that the major carriers for poly-N-acetyllactosamine in human CaCo-2 colonic tumor cells are lamp-1 and lamp-2 and that their poly-N-acetyllactosamine content is decreased after the CaCo-2 cells are differentiated and lose their tumorigenicity (51). These results indicate that the amount of poly-N-acetyllactosamine in lamp-1 and lamp-2 is positively correlated to the tumorigenicity. Our study extended these findings in that we compared two pairs of highly metastatic and poorly metastatic sublines, all derived from a single humen colon cancer specimen. The increased amount of poly-N-acetyllactosamine in lamp-1 and lamp-2 of the highly metastatic cells implies that they are related to tumor progression and the acquisition of the metastatic phenotype.

The present study also revealed that the highly metastatic sublines express more lamp molecules on the cell surface than the lower metastatic sublines. The increased expression of lamp-1 and lamp-2 on the surface of the highly metastatic cells may be related to the metastatic properties, especially as mediators of cell adhesion. In order for tumorigenic cells to establish metastasis, blood-borne tumor cells need to lodge in capillary beds at junctions between endothelial cells (52). Studies from several laboratories suggest that this process can be mediated by interaction between adhesive molecules, selectins, on endothelial cells and the surface of tumorigenic cells (53,54). It has been demonstrated recently that adhesive molecules, named selectins, such as ELAM-1 on endothelium and GMP-140 on platelets bind to sialyl LeX or its related structures on neutrophils and monocytes (6-9, 55). Further- more, several laboratories showed that human epithelial carcinoma cells in situ, in particular those derived from colon, stomach, and breast, express a significant amount of the sialyl LeX and sialyl Le" structures on the cell surface (10-12, 56). This raises the possibility that blood-borne metastatic tumor cells aggregate with platelets in the circulation and succeed in attaching to endothelial cells through the interaction between tumor cell surface sialyl LeX (8) or sialyl Le" (57) structures and selectins present on endothelial cells. The present study revealed, however, that both highly and poorly metastatic tumor cells express almost the same number of sialyl LeX determinants at saturating antibody concentrations, whereas the highly metastatic cells express more of the sialyl Lex determinants with high affinity to the antibody (Table VIII). The results therefore indicate that the role of sialyl LeX is independent of the total number of sialyl LeX structures, if sialyl LeX plays any role in the metastatic differences shown by these cell lines. It is also possible that sialyl LeX determinants on highly metastatic cell lines are recognized more favorably by mouse ELAM-1 because of a more favorable structure in underlying glycans such as polylactosaminoglycans or protein backbone. However, further studies are necessary to determine if the differences in the affinity of anti-sialyl LeX antibody binding can be extended to differences in ELAM-1 binding affinity.

It has been shown that one of the important factors that


enable tumor cells to become invasive is their ability to secrete lysosomal enzymes that can degrade surrounding extracellular matrix (58-60). This release of lysosomal enzymes could be most efficient if intact lysosomes are transported to the cell surface and the lysosomal enzymes exocytosed en masse. In fact, it has recently been shown that cytotoxic T-lymphocytes release lysosomal enzymes by fusing secretory lysosomes with plasma membrane when they attack target cells (61). In addition, we have shown recently that polylactosaminoglycans attached to lamp molecules apparently contribute to the sta- bility of lamps, by protecting the polypeptide moiety from proteolytic digestion in the lumen of lysosomes (28). It is then most likely that secretory lysosomes can reach the cell surface more efficiently in highly metastatic cells and increase their ability to degrade extracellular matrix components and sub- sequent invasion. It will be interesting to determine in future studies if poly-N-acetyllactosamines on cell surface lamps are actually major ligands on metastatic tumor cells recognized by selectins and if more stable secretory lysosomes are present in highly metastatic tumor cells.

Acknowledgments-We thank Dr. Isaiah J. Fidler for providing the colon carcinoma cell lines, Dr. Kentaro Maemura for his assistance in some of the experiments, Dr. 018 Hindsgaul for providing the synthetic substrates, and Henny Bierhuizen for secretarial assistance.

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~ ~~ ~~ , ~~~~ ~~~~

Continued on next page.


SUPPLEMENTAL MATERIAL

TO

LYSOSOMAL MEMBRANE GLYCOPROTEINS IN SUBLINES OF A HUMAN COLON DIFFERENTIAL GLYCOSYLATION AND CELL SURFACE EXPRESSION OF

CANCER EXNlBlTlNG DISTINCT METASTATIC POTENTIALS

BY Osarnu Saltoh, Wet-Chun Wan& Rauiwn Lotan, and Minoru Fukuda

RESULTS

Structuroa 01 Glycopeptldes Bound lo T o m t o Lactln-5.pharoso

S - S e p h a r o s e , the glycopeptide fractions designated - TO analyze the SINclure of complex Asn-linked

as I0 in Fig. 3 were Rret subjected lo endo-pgaladosidase digeslion. Fig. 5 (tight upper pannel) demonstrates lhat intad 10 glycopeptides were scarcely hydrolyzed (6% 01 total radioactivity) by endo- B-galaclosidase whereas aner delucosylation (Fig. 5. right lower panel). lhe amount of released oligoaeccharider is i m a 5 e d 10 32.6% of the total radioactivity. Since lhis increase was not observed aler neuraminidase treatment, the results suggest that fucose residues attached to N- acatylglucosamlne hinder the hydmlysis. This is because endo-b-gaiaclosidase cleaves galaclose linlcspe mwh slower when Kacetylglucosamine is substituted with fucose as follows:

. .

1 0 2 R~Gaigl - t4G1cNAc~l -13Gai~ l~GkNAc~1~

3 f

* 3

Fuml tFucol t

If GkNAc 2 is substkuted with Iucose. lhis poly-N-acalyllanosaminyl st~cture is almost complerely resistant to endo-b-galactosidase (4). Methylation analysis of [~H[-glucosamine-labeled 10 glycopeptides yielded a significant amount 01 6-0-methylglucosamine. supponing lhe above conclusion (Fig. 6A).

The olimsaccharides rewvsrsd in Fractions a and b In Fin. 5 wern subincled 10 OBOW chromatograiph Fig. 7 reveals lhat tractions a c ~ n t a i n s ~ o l i g o ~ ~ h a ~ ~ d e s corresponding 10 NeuNAco2~3~albl-t4(~Fucol~3)GlcNAc~l-r3Galgl~4(Fuc~l~3)GlcNAcS1~3Gal (peak 1 ) . Ne~NA~a2~3Gal8 l -4 fFuca I~3 IGbNAesl~3Gel f m a k 21. N ~ u N A c n 2 ~ 3 G a l n 1 ~ 4 G I ~ N A ~ u l ~ 3 G ~ l

~.~~ ~~ ~ _ ~ _ ~ Galgl-4(Fuml-r3)GlcNACBl-r30al (peak 4). Gaibl-4GlcNA~l~3Gal (peak 5). and GkNAc$l+3Gai (peik 3 ) , ~ F " c ~ l ~ 2 G ' a l ~ i ~ - 4 ( ~ ~ ~ ~ 1 ; j ) G i ~ N A c b l ~ 3 G a l (the leadhi-zhoulder- o l -bak3) ;

desialylmion and defumsylation. the treatmem by endo-ggalaclosidase of I0 glycopeptides yielded the (peak 6). Among those. NeuNAco2~3Galgl-r4GlcNAcQ1~3Gal (peak 3) is the major pmducl. After

ltisacchaIX1e Gdgl-4GkNAcgl~3Gal, and the disaccharide GkNAcgl-3Gal (Fig. 8 A ) . branched oligosacchatide Galgl-4GlcNAc~1+3 (Gal~l-4GIcNAc~I-16)Gal. in addition to

I _ ~ ~ ~ . . .. ".

8 Of I0 glympepMas suppons the above conclusion and a small amount 01 2.4-di-Omelhy1galaclose, 3.4.6-

' . Melhylation analysis of [~H]-gaIacIo~e-IabeIed IB

Iti-Omathylgalaclose and 2,3.4.6-1etra-C-melhylgalact0se was producad in addition lo 2.4.6-lti-O-

amoum of 2.4. 6-tti-C-methylgaladose was decreased with the concomitant increaw 01 2.3,4.6-lelra-O methqdgalaaose Imm intact glycopeptides (Fig. 9A. dosed circles). Aner removal 01 sialic acid, the

palsdose Is present as imernal residues wilhin linear N-acatyllactwmine repeats. About 10% of the methylpalanose (Fig. 9A. open circles, see also Table It). The results indicale that 20-22% 01 (he

galsdos~ residues are in the branch point. R+GIcNAcBl +3(R-GkNAcpl+3)Gal. based on lhe amount of 2,4di.Omethylgaladose (TaMe 111, supporting the above resuks obtained by endo-pgaiaclosidase Ireamem. Similar(y. about 10% of the galaclose residues are substituted wilh 01-2 fucose. based on the amoum of 3.4,6-lri-Omethylgalaclose (Table 11).

To eetimate the told number of Kacetyliaclosamine unim in the IB glycopeplides, the following analysis was camed out. Fiml. methylation analysis of pH]-mannose-labeled glycopeptides was

Ielrssntennary saccharides. but a very minor ponion (74%) of lamp-I glycopeptides consists 01 pedonned. The resuns shown in TaMe 111 indicate thal almost ai1 01 the I0 glycopeptides consin 01

ttianlennary saccharides. When this ianer ponion was taken inlo accounl. I0 giycopeptides from desialylaed lamp-I contained 3.93 moles 01 terminal galaclose (4~0.93 + 310.07). Thus. the total amount of 3,4,6-tti-Omelhylgalaclose and 2,3,4.6-1e1ra-0-methylpalactose should be 3.93 moles. Based on lhis number, each galactose derivative can be Omained (lor example 3.93 x [6O.W0] - 3.4) (TaMe 11). Thus, in lotal. I0 glycopeptides from L4 lamp1 comaln. on average. 5.6 moles of galactose per mole 01 glycopeptide. Similarly, the numbers 01 Nacatyllactwamine units were calculated for other glycopeptides. Based on the amount of 2.4.6-lri-Omelhylgalaclose before and aner desialylation. IB glycopeptide8 01 L4 lamp1 contain, on average, 2.8 moles of sialic acid residues. In fad. the resuits shown In TaMe IV indicate the1 the mabrity of I0 glycopeptides from L4 lamp-1 are of the ttidalyl and dlSiajyl 1orms.

. . acetyllaclDllamine slde chains 01 IB glycopeptides are heavily fucosylated. In order to eslimate the

. The above results suggest that N-

shown in Table 111 and V, the mi0 ot pnl-fucose to [3H]-mannose were determined aner mid hydmlpis Iucose resldues attached 10 N~acelyllactosamine units, the following analyses were carded out. As

or after methylation foliowed by acid hydrolysis. [3H]-fucose is formed by (he melabolic conversion of [3Hl-mannose but the speCiRC actilly 01 pH)fucose is usually lower than the stanins [~H]-mannose. However, the results in Table 111 and V should reWecl the difference in lumse content among different samples; IAl glympeptides are much more fucosylated than other glycopeptides and glycopeptides fmm low metaststlc cells are more fucosylaled than those from highly melaslalic cells. More dellnilive data were omained on methylation analyds of [3H]-pluwsamine-labeled samp(es. In this analysis. 6 . 0 mathylglummine is produced from the Nacetylpiucosamine resldues substituted with fucose at C-3

glumMmine derivatives. The 6-C-melhylglucosamine was diminished aner the glycopeptides were (or C-4). The rewb shown in Fig. SA indicate that 6-Omethylglucosamine constitutes 47% 01 the total

defucosylated, followed by melhylation with the collcomitam increase of 3.6di-Omethyiglucosamine (dam not shown).

indicate that 3.6 moles 01 fuwse residues are attached to N-acetyllaclosamine units. The same Since I0 glycopepUdes of L4 lamp1 comsin 7.7 moles of N.acetyigiucosamine, the resulls

glwopeplides contain. on average, 5.7 moles of Nacetylladosaminyl unb, indicalinp that 63% of the Nawlyllactosamlnyl units are modified by fucose reddues.

Structuro 01 1A2 Glycopeptides Bound to DSA-Agarose

. . .. -e (Fig. IO). The resulm are consislent with the above resulm and

' - I42 glycopeptides could be hydrolyzed almoat to the Same enenl

indicate lhat the majomy of N-acetyigiumsamine reddues, thal are directly aneched to o-mannose residues. are not substiuted wkh fucose. The Paper chmmatwraphy of released oligosacchatid?s revealed thal NeuNAc~-3Gai~l~4GlcNAcgI-r3Gal and Gal$l+4GlcNAc~1+3Gal are the mapr oligosaccharides while N e u N A c o 2 ~ 3 G a l g l ~ 4 ( F u c o l - + 3 ) G l c N A c p l ~ 3 G a l and possibly Fuc~l~2Galgl~4(Fuml-r3)GIcNAcgl~3Gal were also released fmm low mmastaic clone SP (Fig. 1 1 ) . The release 01 GkNAcpl~3Gal from defucosyiated It? glycopeptides (Fig. 80) indicates that 25% 01 poly-Nawtyllsdosaminyl side chains contains three Nacatyllactosamine units.

Structure 01 IA l Glycopeptldes

tomato-Sepharose. and DSA-Agarose. Methylation analysis of [W]-mannose-labeled IA l IAl glycopeptides were unbound to all 01 lhe three lectin affinity columns 01 ConA-Sepharose.

glycopeptides provided 3.4.6-tri-Omelhylmannose in addition to 3.4-dl-Omethyl. 3.6di-Omelhyl. and

tetraamennary saccharides (73.65%) and ltiantennary saccharides (27.35%). Based on these results 2.4-dl-Omelhyimannose (TaMe Ill). The results indicate that IAl glycopeptides are a mixture 01

and lhe results shown in TaMe 11, it can be enimated thm IAl glycopeptides from SP lamp-1 contain 3.7 moles of l en ind gaiaclose (3.1 moles plus 0.6 moles 01 2-linked galadose) and 0.6 moles 01 lmemal

the major dinerenca was noticed in the amount 01 lucose residues anached to N-acetylgiucosamine. Although the above results show that IAl and I42 glycopeptides appear to have similar s I ~ ~ u r e s .

Firsl. intacl IAl glycopeptides were barely susceptible 10 endo-pgalactosidase (Fig. 12). Aner delucosylation, however. IAl glycopeptides released an almost identical amount of oligosaccharides as IA2 glycopeptides (Fig. 12). Among the released OligOsaCCharides, more fucosylated oligosawhatides were released from intact IA l glycopeptides than IA2 glycopeptides (Fig. 11). Finally, [3H].glucosamine-labeled 1Al glycopeptides yielded more of 6-Omethylglucosamine (54% of tolai N. acetylglucosamine residues) than IB or IA2 glycopeptides (Fig. 6C). Since IAl giycopeplides conlaln 6.3 moles of N-acetylglucosamine as lotal. 3.4 moles of N-acatyllaclosaminyi unks is subatiluted with fucose. indicatmg that 79% (3.U4.3 - 0.79) 01 the N-acelyllactosaminyl units are lucosyiated.

galadose. thus 4.3 moles Of galsdOs8 M total (Table 11).

In summary. the majority 01 iAl glycopeptides are telraamennary saccharides wilh or without one N-acetyllaclosamine repeat and these N.acetyllaclosaminyl units are heavily fucosylated. A small proponion (27.35%) 01 iAl glycopeptides are ltianlennary saccharides 01 which N.acetyllactosaminyl unks are also heavily fucosylated.

Glycopeptides IA2 wen not bound to tomato IedinOephamse but bound lo DSA-Agarose column and eluted wifh a mixlure of chiloblose and chitaltiose (Figs. 3 and 4).

-s conaim 01 exdudvely tetraamennary saccharides. Thus ' . The methylation analysis of pH]-mannose-labeled

IA2 glympeptides should contain 4.0 moles 01 terminal Qalaclose residues (unsubsliiuted galaclose plus 2-linked galaclose) (Table 11). It is then possible 10 calculate that 0.6 to 0.9 moles 01 internal g a l a a w wlduas should be present n is nneMnhy that IA2 (and IAl) glycopeptides did not pmduce any 2.4di-OmeIhylpalaclose. indkating thal branched galaawe is absent in lhese glycopeptides (see also Figs. 8B and 96). Thus in total I42 glycopeptides contain 4.6 lo 4.9 moles of galadose residues. The methylation anaiysh oblained before and aner desialylation indicated that the number of sialic acid reslidws in I42 glycopeptides were lound 10 be 2.1 moles to 2.9 moles, which is consiatenl with the

glucosaminslaboled lamp showed that. compared lo IB glycopeptides. an.almwt identical amount of resuils obtained by QAE-Sephadex chromatography (Table IV). Methylation analysis of [W]-

6-Omelhylglucosamins was pmduced fmm IA2 glycopeptides (see Fig. 6). The resub indicate that appmximateiy 2.5 moles of Iuwse is anached to Kacetyllactwmlnyl unks in IN glycopeptidee.

FRACTION

Fig. 4 p . .

The cells were labeled with pH]glucosamine and lamp2 was immunopreapltated as seen in Fig. 28. Glycopeptides were prepared by Pronase digemion and radiolabeled glycopeptides were sequentially applied to leclin-Sephamse columns and analyzed as in Fig. 3. DSA. D. sfrarnonium agglutinin.


CM FROM ORIGIN

500

400

300

2ao

100

2 0 0

200

150

100

so

0 0 5 10 0 5 IO

3w

2w

1 DO

0

6W

4 9

300

150

0

C M FROM ORIGIN Fig. 6 1 3 -

in Fig. 3, were methylaled The resunan1 methylated monosaahandes were separaled by Ihm-layer p H ] ~ l u ~ ~ ~ m i n e - l a b a i e d gtycawphdes from lampt isolated lmm L4alIs . t(sd1onated asshown

Chromatography as desulbed In 'Expenmental Pm~~dures:

A, 8. C and D are L4-IB, IA2, 1A1 end HL-60 lamp1 IE (28) glycopeptides. respectively. The migralion posinons 01 the methylated SUQBD are 1 . 3-Omethyl Namlylgluasamine: 2. 6-Omelhyl N amtylmnhylpluwsamlne; 3. 3.6di-Omethyl N-aeetylrnethylglucosamlne. Under these cond~llons, 4.6-di-Omethyl Nac3tylmelhylpluaosam~ne miorales elmon et tM same pasolion as 3.6-dl-Omethyl N acetylmelhylglucosamine. The radioanlvlty close lo the origin is due to incomplete N.acetylaltan at gluwsamine.

A and C. OllgomCChariaeS Imm SP and L4. resmlvely: Band D. oligosaccharides from HL-60 cells The oliissccharids3 denoted as a in Fig. 5 were separately subisUe3 to paper Chmmatography.

and 0 purlfled oiIgosacEheride. NeuNACd-3GalBl-4GkNAc$l -Sal. respjeethrely.

1. iNsu~AoD2-r3Gal~l~4( iF~~nl~3jGlcNACB~~3Gal~t~4(Fuul l -3~GlcNAc~l~3Gal; 2. NeuNAoD2-r3Gal~l~4(Fucol~3lGlcNA~1~Xjal; 3. NeuNAsa2~Galpl~GlcNAcB1~3Gal:

4. G3b114(Fu~l-r3)GkNAcel-30al: 5. Gal~l4GIcNAcBl~O; 6. GlcNAcpl-r3Gal. F ~ l ~ 2 G a i ~ l ~ 4 ( F ~ m I ~ 3 ) G i c N ~ ~ l ~ 3 G a l ( a s a leading shoulder 01 peak3):

The ml@ral!ons 01 the OllgOBaOCharideS are indicated by numbers as 1011om:

CM FROM ORIGIN

p" Fig 8

A. B and C are Iranionr IBb. IAZb, end IAlb. respeclivelp. which were obtained from Iemp-l The oliposafchandes denoted as Q in Fp. 5, 10. and 12 were wblmed to paper chromatography.

p l~pep l idea 01 L4 mlia. The mlpretton poslllona 01 G a l a l ~ 4 G l c N A c B I ~ 3 ( G ~ ~ l ~ 4 G l c N A c ~ l ~ 6 1 G a l (11. Gdfl114GkNAcplXGal (21. and GkNAcdl-3Gel (31 are mdmted.


L4

200

1 0 0

0 300

I r 0 n B B

L4 afler defucosylatlon r

L4 afler defucosylatlon r I I

C I

FRACTION

50 pel filtration betore 1-0.) and after (-0.) endo-PQalactosidase digestion. The second set 01 p H ~ G a l ~ o s e . l ~ l e d IAl glycopeptides tlom lamp1 mo18cu1es were subjected 10 Sephadex 0.

experimenls was made after removal of (umse and sialic add (denoled as defuwsylallon). 5 i o i 5

CY FROM ORIGIN

Fb.0

PHlgaladose-labeled glycopeplMes Imm L4 cells. tractionaled in a similar way as shown in Fig. 3, were methylated. The resultan1 methylated monosaccharides were separated by thin-layer chromatography as described In 'Experimental Prwedures:

A 18 g1ympeptid.s B IA2 glympeptidw C IA l pty~pepltd~S. Intact glympeptides (-O-). ds~iaed piycopepllde; (&I. me mbralion pdsnldns 01 methylated sugars are as ~OIIOWS: 1 2 4.4

melhyIgalacIose. OmethylpalacIoM; 2. 3 .4 .0 - l r i -Omelhy i~a l~~e; 3, 2.4.6-lri-0methylpaladose: 4, 2,3.4.6~lel;a-O

Lamp-r 0.1 M." GlCN

SP L4 C SM SP L4 SP 14

yklo b u n d 958 94.9 93.2 94.3

4.0 14.2 6.9 12.2 2 6 9.0 5.3 26.4

1 A 2 DSA bund 9.9 27.3 22.1 30.3 IAI OSA unbund 81.9 U.4 64.2 51.8

6.2 28.3 6.5 17.7 68.0 37.3 81.4 46.3

76 8 74.6 93.2 90.4

m i 3.0 3.3 3.6 3.6 1.2 1.4 4.5 5.6

GSri4.l 1.2 1.8 3 2 2.1 22.0 24.0 2.3 4.0

200 1 h A SP L4 C SM SP L4 SP L4

96.3 95.0 94.8 96.5 4.1 16.6 6.5 13.4

IN DSA bund 11.9 30.7 238 30.0 3.3 9.4 70 3 3 2

IA1 DSA unbound 80.3 47.7 64.5 53.1 67 7 33.7 76.9 43.4 8.6 35.0 9.9 15.0

79.6 78.1 938 91.6

z 0 n 2.5 3.2 3.5 2.0 1.2 1.3 4.0 5 1

1.2 1.8 1.7 1.5 19.2 20.6 2.2 3.3

FRACTION

Fig.10 . . 3-

[3H]-galaaose-iabeled IA2 glycopeptides from SP and L4 lamp-l moiecules were subjected 10 Sephadex 0-50 gel filtration before (.O.) and aner (-0.1 endo-8-galaclosidase digeslion. The chromalopraphic condaions are the Same as shown for Fig. 5. The second 881 at experiments was made alter removal 01 lucose and sidic acid (denoted as delumsylalion).

2.4dl-Omethyl 10.5 (06) 9 4 (0.5) 3.4.6-lri-Omemyl 10.0 ( 0 6 ) 7.0 (0.4)

8.3 (0.5) 10.1 (0.6)

2.4.6-WOmehylC 6 2 (0.3) 9 5 (0.5)

2.3.4.6-1mm-Ommh~I 55.8 (3.1) 19.5 (1.1) 70.5 13.91 19.9 lI.11 23.7 (1.3) 64.1 (3.5) 15.0 10.9) 60.5 (3.4)

I rmn.9 - E%L-l 16.0 (1.0) 12.6 (0.81 3.4.6-ld-Omemyi 2.4.8-ld-Omemyl 2.3.4,6-letra-OmethyI

9.6 (0.5) 7.6 (0.41 13.1 (0.8) 9.9 (0.6) 10.5 10.6) 116 (0.71 59.6 (3.6) 20.5 (1.2) 66.6 13.8) 22 3 (1 31 11.3 (0.7) 57.0 (3.4) 12.2 (0.71 58.5 (35)

E%%memyi 10.0 (0.5) 10.4 (0.5) 9.2 (0.4) 8.1 (0.4) 2.4.&1d-Onlethvi 2.3,4,6-tetm-0memy1

64.3 (3.2) 18.7 (0.9) 76.3 (3.6) 14.0 10.71 25.7 (1.3) 70.9 (3.5) 14.5 (0.7) 77.9 (3.6)

IAI Qtycopeptldes Lamn-l

I rma.2

CY FROM ORIGIN

Fip. 11 1 ..

Olipoaacchsrides a shown in Fig. IO and 12 were subjected 10 paper chromatography.

A, IA2 glycopeptides tmm S P E. IA2 QlyqeplMes from L4; C. IAl glympeptides from Sp: D, IAl ~Iympeplldes from L4. The oligomaride nandards am the same as shown in the lepend of Fig. 7.


Lamp-1 Tomalo OSA DSA

SP

bound bound unbound bound bound unbund IB lA2 IA1 I0 IA2 IA1

Tomato OSA DSA L4

"Qsa 3.4 (or3.6pl-amethyla 1.92 2.00 I 70 1.93 2.00 1.65 2.Cdi.Ommtyl 3.4.6.bi-0msmy(

1 1 1 0.08 - 0.30 0.07 - 0.35

1 1 1

Evcpaa 2.3.4-m-amemylb 1.0 0.44 2.99 0.48 0.21 1.25

S P Tomato DSA DSA

I0 w v\1 I0 W2 1A1

L4 Tomato DSA DSA

Lamp-2 bound bound unbound bound bound unbound

Lamp-1 0 7 . P 12.2 10.6 1 13.9 13.3 124

4.7 2.2 9.7 9.6 3.4 11.3

2 32.0 31.2 29.6 32.2 22.8 33.8 3 352 349 300 44.7 59.6 3 L . 2 4 9.7 6.5 9.9 6.7 10.1 6.3 5 and higher 2.3 1.9 7.4 2.0 2.0 4.7

x x

Lamp-? 0 6.0 10.5 123 3.8 3.8 10.9 1 101 11 6 12.7 6.6 4.3 11.1 2 30.3 27.9 29.5 30.9 20.9 27.9 3 40 0 35.9 3 . 5 47.8 53.6 33.0 4 10.7 11.3 6.0 8.7 14.9 12.0 5 and hiaher 2.8 2.8 3.1 2.2 2.4 4.0

SP L4

Con AI I0 W2 IA1 Con AI I0 IAZ 1A1

Lamp-1 3.06 1.21 1.10 3.52 1.44 0.92 0 78 2.02

Lamp-2 3.01 1.19 104 3.39 1.39 0.67 0 6 5 1.99

the of vol. 267, no. of 15, 5700-5711,1992 1992 printed in ... · 1 and lamp-2 antibodies were...

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