THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1986 by The American Society of Biological Chemists, Inc.

Val. 261, No. 27, Issue of September 25, pp. 12787-12795,1986 Printed in U.S.A.

Structural Variations of 0-Linked Oligosaccharides Present in Leukosialin Isolated from Erythroid, Myeloid, and T-Lymphoid Cell Lines*

(Received for publication, November 15, 1985)

Sven R. Carlsson, Hiroshi SasakiS, and Minoru FukudaP From the Cancer Research Center, La Jolla Center Research Foundation, La Jolla, California 92037

Structures of 0-linked oligosaccharides of leukosi- alin isolated from K562 erythroid, HL-60 promyelo- cytic, and HSB-2 T-lymphoid cell lines were examined. Leukosialin was isolated by specific immunoprecipi- tation from cells which were metabolically labeled with [3H]glucosamine, and glycopeptides were isolated after Pronase digestion. 0-Linked oligosaccharides were released by alkaline borohydride treatment, and the structures of purified oligosaccharides were eluci- dated by specific exoglycosidase digestion, Smith deg- radation, and methylation anaylsis. Oligosaccharides from K562 cells were found to be GalNAcOH, Gal@1+ 3GalNAcOH, NeuNAca2+6GalNAcOH, NeuNAcaZ+ 3Galb1+3GalNAcOH, Gal/31+3(NeuNAcaZ+G)Gal- NAcOH, and NeuNAca2+3Gav1+3(NeuNAcaZ+ 6)GalNAcOH. On the other hand, oligosaccharides from HL-60 and HSB-2 cells were found to be Neu- NAca2+3Galj31+3GalNAcOH, NeuNAca2+3Gavl+ 4GlcNAc~1+6(Ga~1+3)GalNAcOH, Gal@1+4Glc- NAc@1+6(NeuNAca2+3Gal@1+3)GalNAcOH, and NeuNAca2+3Gal~1+4GlcNAc@1+6(NeuNAc~2+3- Galj31+3)GalNAcOH. These results clearly indicate that leukosialin can be differently glycosylated with 0-linked chains, and each erythroid or myeloid (and T-lymphoid) cell line expresses a characteristic set of 0-linked oligosaccharides which differ in core struc- tures as well as in sialylation.

~ ~~~ ~

In the preceding paper (I), we showed that various leukemic cell lines express a family of sialoglycoproteins which are closely related to each other. Although these glycoproteins isolated from different cell lines show significant differences in molecular weight, as estimated by SDS-polyacrylamide gel electrophoresis, their polypeptide portions appear to be very similar or the same, based on their size and reactivity with antibodies. These glycoproteins, termed leukosialin, were shown to contain a large number of 0-linked oligosaccharides and one asparagine-linked oligosaccharide/molecule (1).

In this study, we present the evidence that the apparent differences in molecular weight of leukosialin in different cell lines are due to variations in 0-linked oligosaccharides; the

* This work was supported by Grants R01 CA 33895 and CA 33000 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 “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ On leave of absence from the Central Research Institute, Chugai Pharmaceutical Company, Tokyo, Japan.

5 To whom correspondence should be addressed La Jolla Cancer

92037. Research Foundation, 10901 North Torrey Pines Rd., La Jolla, CA

’ The abbreviations used is: SDS, sodium dodecyl sulfate.

structures of 0-linked oligosaccharides differ significantly depending on the cell lines from which the glycoproteins were isolated.

EXPERIMENTAL PROCEDURES

Isolation of [3H]Glucosamine-labeled Leukosialin from K562, HL- 60, and HSB-2 Cells-The cell lines were cultured in RPMI 1640 tissue culture medium supplemented with 2 mM glutamine and 10% fetal calf serum. For metabolic labeling with [3H]glucosamine, cells were harvested and cultured in medium containing 1% of the normal content of glucose supplemented with [3H]glucosamine at 4pCilml. ~-[6-~H]Glucosamine (32.9 Ci/mmol) was purchased from New Eng- land Nuclear. Labeling was conducted for 24 h, after which cells were harvested and washed with phosphate-buffered saline. The metabol- ically labeled cell pellets were lysed by the addition of 20 volumes of cell lysis buffer (phosphate-buffered saline containing 0.5% nonidet P-40, 1 mM phenylmethylsulfonyl fluoride). After occasional vortex mixing at 4 “C for 30 min, the mixture was centrifuged at 8000 X g for 10 min at 4 “C. Leukosialin was isolated by immunoprecipitation with specific anti-leukosialin antiserum as described in the preceding paper. (1). Similarly, [3H]galactose-labeled leukosialin was obtained by metabolic labeling with [3H]galactose (44.1 Ci/mmol) followed by immunoprecipitation.

Preparation of Glycopeptides from Leukosialin-Glycopeptides were prepared from the immunoprecipitates by Pronase digestion at 60 “C for 48 h. The Pronase was preincubated for 1 h at 37 “C to inactivate potential contaminating glycosidases. Twenty-five pg of Pronase was added at 0 and 24 h to each immunoprecipitate which was suspended in 0.5 ml of 0.1 M Tris-HC1, pH 7.8, containing 1 ml of CaCl, and a few drops of toluene.

Preparation of 0-Linked Oligosaccharides from Leukosialin Glyco- peptides-The Pronase-digested material was applied to a column (1.0 X 110 cm) of Sephadex G-50 (superfine) equilibrated with 0.1 M NH4HC03. The flow was 6 ml/h, and each fraction contained 1.1 ml. The fractions containing radioactive glycopeptides were pooled and lyophilized. Each glycopeptide fraction was then treated with 0.05 M NaOH in the presence of 0.4 M NaBH, at 45 “C for 16 h (2). The pH of the samples was then neutralized by adding methanol containing acetic acid and lyophilized after addition of water. After dissolving in 0.1 M NH4HC03 and centrifugation, the supernatant was applied to the same column.

Fractionation of Oligosaccharides by QAE-Sephndex Column Chro- matography-Oligosaccharide fractions obtained after Sephadex G- 50 were lyophilized, dissolved in 2 mM Tris-HC1 buffer, pH 8.0, and applied to a column (0.4 X 17 cm) of QAE-Sephadex A-25 equilibrated with 2 mM Tris-HC1 buffer, pH 8.0. After washing with 5 ml of the same buffer, the elution was performed by a linear gradient from 2 mM Tris-HC1, pH 8.0, to 50 mM sodium phosphate buffer, pH 7.4 (each 16 ml). The flow rate was 5 ml/h, and fractions (0.5 ml) were collected. After the gradient elution, the column was washed with 0.2 M sodium phosphate buffer, pH 7.4, in order to elute highly charged materials.

Bio-Gel P-2 Gel Filtration of Oligosaccharides-Oligosaccharide fractions obtained after Sephadex G-50 gel filtration followed by QAE-Sephadex column chromatography were applied to a column (1.0 X 110 cm) of Bio-Gel P-2 (200-400 mesh). The column was equilibrated with 0.1 M NH4HCO3, and the flow rate was 6 ml/h and each fraction contained 1 ml.

Glycosidase Treatments-Saccharides were digested with jack bean

12787

12788 Structural Variations of 0-Linked Oligosaccharides

8-galactosidase (0.125 unit) and @-N-acetylglucosaminidase (0.125 unit) in 50 mM sodium citrate buffer, pH 4.3, in a final volume of 50 pl. 0ligosacci:arides were also digested with diplococcal @-galactosid- ase (IO milliunits) in 50 pI of 0.1 M sodium acetat.e buffer, pH 5.8, at 37 "C for 24 h. Treatment with Escherichia coli @-galactosidase (20 units) was done in 80 p1 of 50 mM sodium phosphate buffer, pH 7.3, containing 4 mM MgC12. In order to inhibit a possible con taminahg activity of @-N-acetylglucosaminidase, 8 mM (final concentration) N- acetylglucosamine was added to the incubation mixture of E. coli @- galactosidase. Oligosaccharides were desialylated either with mild acid hydrolysis in 0.02 N HCI at 80 "C for 1 h or Vibrio cholera neuraminidase (20 milliunits) in 60 +I of 20 mM sodium acetate buffer, pH 5.8, containing 2 mM CaCI2, a t 37 "C for 24 h. (u2+3- Linked sialic acid was specifically removed by Newcastle disease virus neuraminidase as described (3.4). Newcastle disease virus neuramin- idase was kindly donat.ed by Dr. Paulson, UCLA School of Medicine. Diplococcal @-galactosidase (5,6) was kindly donated by Dr. Michiko N. Fukuda of this institute. All the other glycosidases were purchased from Sigma except V. cholera neuraminidase which was from Behring Diagnostics.

Paper Chromatography-Paper chromatography was carried out in a descending manner using the solvent system butanol/pyridine/ water (6:4:3) for 36 h. The sample lanes were cut into 1-cm sections, and radioactive spots were detected by liquid scintillation counting. A small amount of water was added to the paper before the addition of the scintillator as described (7). Standard monosaccharides and oligosaccharides were located by the silver nitrate staining procedure (8). Standard N-acetylgalactosaminitol was prepared by reduction of N-acetylgalactosamine in the presence of NaB[:'H],.

Periodate Oxidation of0li~osaccharides-Oligosaccharides were ox- idized with 200 pI of 50 mM NaIO, in 50 mM sodium acetate buffer, pH 4.5. The solutions were kept in the dark at 4 "C for 20 h. The reaction was terminated by adding about 30 pl of glycerol at room temperature for 1 h. The samples were then reduced by sodium borohydride at room temperature for 4 h and applied to a column of Bio-Gel P-2. The periodate-oxidized fractions containing oligosac- charides, detected by radioactivity, were pooled and lyophilized. The periodate-oxidized borohydride-reduced samples were then hydro- lyzed in 0.02 N HCI a t 80 "C for 1 h. The hydrolyzed sample was neutralized by adding 0.1 M Tris-HCI buffer, pH 8.5, and subjected to the same Bio-Gel P-2 filtration.

Methylation Analysis-Oligosaccharide fractions were methylated by the method of Hakomori (9) with a modification as described (10). The methylated oligosaccharides were hydrolyzed with 0.5 N H2S0, in 90% acetic acid a t 80 "C for 4 h. The hydrolysate was neutralized as described (10) and dissolved in chloroform/water (1:l) suspension. The chloroform layers, which contained methylated monosaccha- rides, were washed three times wit,h water and stored as frozen before use. The methylated monosaccharides were separated by thin-layer chromatography on plates of Silica Gel G using the solvent system acetone/water/concentratedammonia (v/v, 25031.5) (11, 12). Stand- ard methylated monosaccharides were obtained from NeuNAcru2- 3Gal@l+3GalNAcOH and NeuNAccu2+3GalB1-+3(NeuNAcru2+ 6)GalNAcOH, which were isolated by alkaline borohydride treatment of fetuin glycopeptides (13) in the presence of NaB[:'H],. They were also produced from NeuNAccu24GalNAcOH which was similarly isolated from bovine suhmaxillary mucin (Sigma) (14). 3,6-Di-0- methyl N-methylglucosamine was produced from [:'H]glucosamine- labeled polylactosaminoglycans from PA-1 cell (15).

RESULT?

Leukosialin Preparations Obtained from ['H]Glucosarnine- labeled K562, HL-60, and HSB-2 Cells-Fig. 1 shows the fluorogram of SDS-polyacrylamide gel electrophoresis of leu- kosialin obtained by immunoprecipitation of [:'H]glucosa- mine-labeled cells. Leukosialin prepared from HL-60 and

' Portions of this paper (including part of "Results" and Figs. 2-4 and 6-11) 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 available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Doc- ument No. 85M-3762, cite the authors, and include a check or money order for $8.40 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

lysate i.p.

116- w

66.2-

FIG. 1. SDS-polyacrylamide gel electrophoresis of [3H]glu- cosamine-labeled cell lysate and leukosialin. Cells were labeled with [3H]glucosamine, and aliquots of the lysates (lysate) and im- munoprecipitates with anti-leukosialin antibodies (i.p.) were analyzed by SDS-polyacrylamide gel electrophoresis followed by fluorography.

HSB-2 showed a homogenous single band, whereas the gly- coprotein from K562 showed an additional lower faint band. This band of lower molecular weight was due to proteolytic degradation since this band was increased in the absence of protease inhibitor. Since these isolated samples showed no contaminants, the immunoprecipitates were directly used without further purification.

Isolation of 0-Linked Oligosaccharides from /:'H]Glucosa- mine-labeled Leukosialins-The [:'H]glucosamine-labeled leu- kosialins were digested by Pronase, and the digests were applied to a column of Sephadex G-50. As shown in Fig. 2, glycopeptides from these leukosialins afforded two peaks; one eluted at the void volume (Peak I ) and another eluted as a broad peak (Peak 11). All of these glycopeptides were larger than fetuin glycopept,ides ( M , = 3,100). Peak I and peak I1 from both K562 and HL-60 cells were examined by SDS- polyacrylamide gel electrophoresis followed by fluorography. Interestingly, peak I from each cell line showed heterogenous glycopeptides with apparent molecular weights of approxi- mately 20,000-40,000 (from K562) and of 30,000-50,000 (from HL-60), confirming that leukosialin has a large domain which is heavily glycosylated by 0-linked oligosaccharides (see also the preceding paper ( I ) ) . Apparently, peak I1 did not contain such large glycopeptides. The two peaks were separately sub- jected to alkaline borohydride treatment and applied to the same column of Sephadex G-50. Both Peaks I and I1 from K562 cells provided three radioactivity peaks which eluted between the positions where N-acetylglucosamine and IgG oliogosaccharide eluted (Fig. 3). Peaks I and I1 from HSB-2 cells provided t.wo and three radioactive peaks, respectively. Peaks I and I1 from HL-60 cells, on the other hand, provided

Struc tura l Variations of O-Linked Oligosaccharides

: : 4 3 2

;“ j ; 7 7 * 8 : +

HSE-2-8 HSB-2-C - 0.4

- 30 4 0 50 00 70 80 30 40 50 80 70 80

FRACTION NUMBER FIG. 5. Bio-Gel P-2 gel filtration of oligosaccharides separated by QAE-Sephadex column chroma-

tography. Oligosaccharides B and C, which were separated by QAE-Sephadex column chromatography as shown in Fig. 4, were subjected to Bio-Gel P-2 gel filtration before (dotted line) and after (solid line) desialylation. The K562-D oligosaccharide fraction was directly applied after Sephadex G-50 gel filtration (Fig. 3). The HL-60-D and HSB-2-D fractions did not show any distinct radioactive peaks. The oligosaccharides were pooled as indicated by horizontal bars and 4 , 3 , 2 , and I correspond to the elution positions of tetrasaccharide, trisaccharide, disaccharide, and N-acetylgalactosaminitol. Open vertical arrows indicate the elution position of N-acetylneuraminic acid, which was confirmed by its binding to a QAE-Sephadex column. Closed uertical arrows from left to right indicate the elution positions of bovine serum albumin (void volume, Vo), Gal~l4GIcNAc~l+6(GaI~l~3)GalNAcOH, Gal~1+4GlcNAcB1+3Gal, GalBl+BGalNAcOH, and N-acetylgalactosaminitol. Details for chromatographic con-

12789

ditions are described under “Experimental Procedures.”

two radioactive peaks after alkaline borohydride treatment. The molecular weights of the two larger radioactive peaks from HL-60 and HSB-2 leukosialins were slightly larger than the corresponding two peaks observed in K562 leukosialin. Radioactive peaks from the same cell lines that eluted at the same positions (for example, fraction B in K562 I and fraction B in K562 11) were combined and subjected to QAE-Sephadex column chromatography (see below). Fraction A .from each column was subjected to hydrazinolysis and reapplied to the same column of Sephadex G-50. This treatment converted the radioactivity in Fraction A into radioactive peaks of various sizes, but no distinct peaks were obtained (data not shown). This material was not investigated further.

Purification of Oligosaccharides by QAE-Sephadex Column Chromatography and Bio-Gel P-2 Gel Filtration-Fraction B and fraction C from K562, HL-60, and HSB-2 leukosialin were then subjected to QAE-Sephadex column chromatogra- phy as shown in Fig. 4. Fraction B from three cell lines eluted at positions where disialo-oligosaccharides elute, whereas Fraction C eluted in a range of monosialo-oligosaccharides. It was also apparent that oligosaccharides from K562 leukosialin eluted slightly later than the corresponding oligosaccharides from HL-60 or HSB-2 leukosialin. The major oligosaccharide fractions obtained after QAE-Sephadex column chromatog- raphy (designated by horizontal bars) were then subjected to Bio-Gel P-2 gel filtration before and after desialylation. Disialo-oligosaccharides of K562 leukosialin produced a

disaccharide after desialylation (the solid line in Fig. 5, K562- B) together with free sialic acid, the position of which is indicated by an open vertical arrow. Monosialo-oligosaccha- rides of K562 leukosialin produced disaccharide and mono- saccharide after desialylation (Fig. 5, K562-C). In addition, oligosaccharides of the lowest molecular weight (fraction D in Fig. 3, K562-I and K562-11) were found to contain a neutral

disaccharide and N-acetylhexosaminitol (Fig. 5, K562-D). Disialosyloligosaccharides from HL-60 and HSB-2 cells

produced a tetrasaccharide after desialylation, whereas mon- osialyloligosaccharides from the same glycoproteins produced a tetrasaccharide, trisaccharide, and disaccharide after desi- alylation (Fig. 5). Structures of each separated oligosaccharide were elucidated by exoglycosidase treatment, chromium triox- ide oxidation, Smith degradation, and methylation analysis as described in the “Miniprint” section.

Structures of Oligosaccharides Obtained from K562 Leuko- sialin-The major oligosaccharides from K562 leukosialin are monosialylated trisaccharides (CIII), and their structures were elucidated to be NeuNAca2+3Galpl-+3GalNAcOH and Gal@l+3(NeuNAca2-+6)GalNAcOH (see the “Miniprint” section). The second major oligosaccharide (CII) was found to be NeuNAca24GalNAcOH. The disialylated tetrasaccha- ride (BIV) was elucidated to be NeuNAccu2+3Galpl+ 3(NeuNAca2+6)GalNAcOH. In addition, neutral oligosac- charides, Galpl-3GalNAcOH (D2) and GalNAcOH (Dl), were isolated (see also Table I).

Structures of Oligosaccharides Obtained from HL-60 and HSB-2 Leuhsialin-The oligosaccharides obtained from HL- 60 and HSB-2 are essentially the same. The major oligosac- charide is disialylated hexasaccharide (BVI), and its structure was elucidated to be NeuNAca2+3Galfil+3(NeuNAc~t2+ 3Galpl“*4GlcNAc~1-+6)GalNAcOH. The second major oli- gosaccharide is tentatively assigned to be NeuNAa2- 3Gal~l+4GlcNAc~l-+6(3-deoxy)GalNAcOH. The minor ol- igosaccharides were found to be NeuNAca2+3Galpl-, 4GlcNAc~l4(Gal~l-+3)GalNAcOH, Galp l4GlcNAcpl+ 6(NeuNAca2-*3Gal~1+3)GalNAcOH, and NeuNAca2- 3Galpl-3GalNAcOH (see Table I). HSB-2 leukosialin ap- pears to contain more NeuNAca2~3Galpl~3GalNAcOH than HL-60 leukosialin (see “Miniprint”).

12790 Structural Variations of 0-Linked Oligosaccharides

TABLE I Proposed structures of 0-linked oligosaccharides attached to

leukosialin from K562, HL-60, and HSB-2 cells Oligosaccharides were isolated bv alkaline borohvdride treatment.

Oligosaccharides

GalNAcOH

GalP1-3 GalNAcOH

NeuNAca2 I

6 GalNAcOH

NeuNAca2 I

6 GalP1-3GalNAcOH

NeuNAca2-3Gal~l-3GalNAcOH

NeuNAca2 I

6 NeuNAca2-+3Galpl-+3 GalNAcOH

NeuNAccu2-3Gal~14G1cNAc/31 I

6 GalP1+3GalNAcOH

Gal/31+4GlcNAc~l I

6 NeuNAca2-3Ga1(31-3GalNAcOH

NeuNAcot2-3Gal~l4G1cNAc~l I

6 NeuNAca2-3Gal81+3GalNAcOH

~

+ +

+

+++ +++

++

-

-

-

K562 HL-601 -

+++

FIG. 12. Postulated scheme for biosynthesis of leukosialin-attached 0-linked oligosaccharides in K562, HL-60, and HSB-2 cells. The biosyn- thetic steps were hypothetically con- structed according to Beyer et al. (22), Brockhausen et al. (21), and Williams et al. (32). Enzyme 1, Gal(31-3GalNAc- R (GlcNAc to GalNAc) P6-N-acetyl glucosaminyl transferase; enzyme 2, Gal (31-3GalNAc-R ( s i a l i c a c i d t o Gal) ad-sialyl transferase; enzyme 3, GalNAc-R a6-sialyl transferase. K562, HL-60, and HSB-2 cells represent eryth- roid, myeloid, and T-lymphoid cells, respectively. HL-60 and HSB-2 leukosi- alins contain almost the same sets of 0- linked oligosaccharides (see also “Dis- cussion’’).

DISCUSSION

This paper established the structures of 0-linked oligosac- charides attached to leukosialin from three cell lines, K562, HL-60, and HSB-2. The studies clearly indicate that each erythroid or myeloid (and T-lymphoid) cell line expresses a characteristic set of 0-linked oligosaccharides which differ in core structures as well as in sialylation. Leukosialin from HL- 60, which has the largest apparent molecular weight, contains more complex 0-linked oligosaccharides than leukosialin from K562, which has a smaller apparent molecular weight. Leu- kosialin from HSB-2 cells appears to have oligosaccharides which are less sialylated than those from HL-60 leukosialin, as judged from the chromatogram of Sephadex G-50 gel fil- tration (Fig. 3). This may be the reason for HSB-2 leukosialin showing a slightly smaller molecular weight than HL-60 leu- kosialin although both contain the same sets of oligosaccha- rides. I t should also be considered that some of the differences in molecular weight may be due to differences in the number of oligosaccharides attached to the polypeptide backbone. Above all, it is apparent that differences in 0-glycosylation cause such a significant effect on protein migration during SDS-polyacrylamide gel electrophoresis, and it has been dif- ficult to correlate these glycoproteins to each other solely by this method (20) unless the specific antibodies are used as shown in the present study.

Although leukosialin contains one N-linked chain, the amount of N-linked carbohydrate was very small compared to 0-linked chains since N-linked carbohydrate was not de- tected when the cells were labeled by [3H]glucosamine. The incorporation of mannose into leukosialin was also too little to permit analysis of the oligosaccharides (data not shown). Further studies will be necessary to elucidate the structure of N-linked oligosaccharides.

I t is noteworthy that HL-60 leukosialin and K562 leukosi- alin have distinctly different 0-linked oligosaccharides. In HL-60 cells, the Galpl-3GalNAc core stucture is further

NeuNAcaZ

0 ‘ 6 GalNAca1.R GalNAca1.R

NeuNAca2.

0 \ Gal81+3GalNAcal+R Gal61+3GalNAcal*R

6

Gal81+4GlcNAcBl NeuNAca2 \ \ 6 6

Gal81+3GalNAcal+R NeuNAca2+3Gal81+3GalNAcal+R

+@ NeuNAca2+3Gal81+4GlcNAc8l

\ 6

NeuNAca2*3Gal61+3Ga1NAcal*R

Myeloid Erythroid

Structural Variations of 0-Linked Oligosaccharides 12791

elongated by a Galpl+3GalNAc(GlcNAc to GalNAc)P6-N- acetylglucosaminyl transferase (Enzyme 1 in Fig. 12) to form GlcNAc~1-+6(Gal~l+3)GalNAc, core 2 (see Ref. 21). This branching is further followed by galactosylation and sial- ylation to form NeuNAca2-3Gal@l4GlcNAc@l+ 6(NeuNAca2+3Gal@1+3)GalNAc (see Fig. 12). In K562 er- ythroleukemic cells, the key enzyme 1 may be missing so that Galpl+3GalNAc is not branched further by N-acetylgluco- samine. Instead, it is sialylated by a &galactoside a2-3 sialyltransferase (enzyme 2 in Fig. 12) followed by an a-N- acetylgalactosaminide a 2 4 sialyl transferase (enzyme 3 in Fig. 12). Alternatively, GalNAc or Galpl-3GalNAc is sialy- lated by enzyme 3 to form NeuNAca24GalNAc or GalPl+ 3(NeuNAca24)GalNAc (22). This enzyme 3, however, may be missing in HL-60 cells which represent myeloid cells. These results suggest that each cell type has a characteristic set of glycosyltransferases which form 0-linked oligosaccharides.

The present study did not reveal the differences in struc- tures between HL-60 and HSB-2 oligosaccharides. However, it is likely that HL-60 leukosialin has oligosaccharides as minor components which have G a l ~ l 4 ( F u c a 1 ~ 3 ) G l c N A c terminals since granulocytes of chronic myelogenous leukemia contain such a structure in 0-linked oligosaccharides (see the following paper (23)). HSB-2 leukosialin is expected to lack this structure since only myeloid cells are reactive with monoclonal antibodies which recognize Galpl-A(Fucal+ 3)GlcNAc structures (for review, see Ref. 24).

It has been shown that human erythrocyte glycophorin contains NeuNAca2+3Gal~l-3(NeuNAca24)GalNAc as a major 0-linked oligosaccharide (25). In the following paper (23), we have shown that the major 0-linked oligosaccha- rides in granulocytic cells are kNeuNAca2+3Galpl+ 4GlcNAc@l~(fNeuNAca2+3Gal~1+3)GalNAc. The 0- linked oligosaccharides attached to K562 leukosialin are mainly comprised of NeuNAca2+3Gal/31+3(NeuNAca2+ 6)GalNAc and its monosialylated form, NeuNAca2- 3Galpl-3GalNAc or Gal~l+3(NeuNAca24)GalNAc (see Table I). These results are consistent with the idea that K562 leukosialin expresses 0-linked oligosaccharides of the eryth- roid type but not of the granulocyte (myeloid) type.

Our results also indicate that 0-linked oligosaccharides in K562 cells are slightly different from those on mature eryth- rocytes; the former are comprised of a significant amount of the monosialylated form, whereas the latter contain disialy- lated tetrasaccharide as a major component (25). Thus, it appears that 0-linked oligosaccharides on K562 cells are less sialylated than those on mature erythrocytes. This evidence may be related to the recent observation that immature eryth- roid cells lack MN blood group activity although they express glycophorin A (26). By isolating and analyzing cDNA for glycophorin A, it has been shown recently that the peptide sequence of glycophorin A in K562 cells is identical to that of erythrocyte glycophorin A (27). It is known that the expres- sion of blood group MN activities is heavily dependent on the sialic acid residues of 0-linked oligosaccharides in addition to peptide sequence (28, 29). Therefore, it is likely that MN antigenic activities are not expressed well on K562 cells and immature erythroid cells because of incomplete sialylation of their oligosaccharides attached to glycophorin A.

The present study clearly indicates that different cells (erythroid uersus myeloid) express distinct 0-linked oligosac- charides which are specific to each cell type. It has been shown that different species have different 0-linked carbo- hydrates in the submaxillary glands (14, 30, 31). The present

study showed that cells derived from the same stem cell but in different lineages have distinctly different sets of 0-linked oligosaccharides. Since 0-linked oligosaccharides frequently express unique antigenic structures, it is now possible to evaluate these oligosaccharides as cell lineage specific mark- ers.

Acknowledgments-We thank Drs. James C. Paulson and Michiko N. Fukuda for donating specific glycosidases, Priya Ramsamooj for technical assistance in the early stages of this work, Monique Lauf- fenburger for assistance in the later stages, and Anna Steve and Diana Lowe for secretarial assistance.

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16. Finne, J. (1975) Biochim. Biophys. Acta 412,317-325 17. Van den Eijnden, D. H., Evans, N. A., Codington, J. F., Reinhold,

V.. Silber, C., and Jeanloz. R. W. (1979) J. Biol. Chem. 254.

259,4782-4791

318-322

12528-12535

5704-5717

Bwl. Chem. 260,6623-6631

12153-12159 . .

18. Chandrasekaran. E. V.. Mendicino. A.. Garver. F. A.. and Men- I ~ ~~~

dicino, J. (1981) J. Biol. Chem. 266,’1549-1555

and Liao, J. (1984) J. Bwl. Chem. 2 5 9 , 7178-7186

Acad. Sci. U. S. A. 78, 6299-6303

Biochemistry 24,1866-1874

E., and Hill, R. L. (1979) J. Biol. Chem. 254, 12531-12541

Bwl. Chem. 261.12796-12806

19. Wu, A. M., Kabat, E. A., Nilsson, B., Zopf, D. A., Gruezo, F. G.,

20. Fukuda, M., Koeffler, H. P., and Minowada, J. (1984) Proc. Natl.

21. Brockhausen, I., Matta, K. L., Orr, J., and Schachter, H. (1985)

22. Beyer, T., Rearick, J. I., Paulson, J. C., Prieels, J.-P., Sadler, J.

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24. 25.

26.

27.

28.

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32.

Fukuda, M. (1985) kiochim. Biophys. Acta 780,119-150 Thomas, D. B., and Winzler, R. J. (1969) J . Biol. Chem. 2 4 4 ,

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66,233-236 Siebert, P. D., and Fukuda, M. (1986) Proc. Natl. Acad. Sci. U.

Springer, G. F., and Ansell, N. J. (1958) Proc. Natl. Acad. Sci. U.

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12792 Structural Variations of 0-Linked Oligosaccharides SUPPLEMEMTAL MTERlAL

TO 5TRUCTURAL VARIATIONS OF 0-LINKED OLIGDSACCHARIDES PRESENT 1H

LEUKDSIALIN ISOLATED FRM ERVTHRDID, MYELOID. AND 1-LYHPHIID CELL LINES

BY

Sven 1. Carlrron. Hiroshi Sasaki , and Minoru Fukuda

St ruc tures O f O l igoracchander fro7 1562 Leukos ia l in

Meutral Oli osdcchdrides - K562-D f l a c t i o n produced d isacchandc (K562-02) and mooneraccharide IR562-DlJ. h 2 - D 1 was i d e n t i f i e d t o be N-acetylgalactoraninit~l by paper Chmmtogrdphy (Fig. 6A). 1562-02 was p a r t i a l l y s u s c e p t i b l e to E-galactor idare. y ie ld ing H-acetylgalact~raainitai (Fig. 7A). S i n c e t h e y i e l d o f N-acetylgalactoraminitol was IW. t h e d l s a c i h a i i d e was subjected t o C h r a n i m t r i o x i d e treatment a s described by F inne (16) . A f te r th is t rea tment . the radio- act ive spot detected ran faster than the d i5acchar ide on paper thranatogrdphy (data not shown), c o n f i m i n g 8 - l i n k a g e Of g a l l c t o 5 C . A f t e r p e m t h y l a t i C I n , K562-02 yielded tetra-0-methyl N- a e t h y l g a l a c t a r a ~ i n i t o l wh ich apparent ly Cmlgra ted w i th 1.4.5.6-tetra-O-nethyl N-methylgalacta- ram in i to l (F ig . EA). These resu l t s i nd i ca te t he f a l l ow ing s t ruc tu re f o r K562-02.

Ga1E1*36a1NA~OH

Monosia ly la ted Ol rgoracchander - K562-C f rac t i on y ie lded d l racchar ide 2 and monosaccharide I after d e r i a l y l a t i o n (fTg. 51. The structures of those neutl.1 ol igosaccharides were estab l i shed to be Ga181+3GalNl\cOH and GaIMcOH i n the sane way dl d e r c n b e d above. As in tac t o l igoraccha-

K562-CII . respect ively. There two oligosaccharides were p a r t i a l l y Separated by Bia-Gel P-2 gel r i d e r . one I i a 1 1 c a c i d i s l l n k e d to these ol lgoracchar ider and they were t e m d K562-CIII and

d isacchar ide bu t no N-ace ty l ga lac to rm in i to1 was re leased (Fig. 9Al . Honor ia ly lated f i l t r a t i o n ( F i g . 5 ) . Af te r 02-3 r p e r i f i c n e u r m i n l d a s e t r e a m n t . K562-C f rac t ion p rov ided

o l igOsdCChwide f rac t ion (Frac t ions 40-45 i n F i g . 9A) was digested again by 2-3 r p e c ? f l c neura- minidase but no fur ther degldddt iDn war detected. UonoI id ly la ted Ol igoIaCChwlde f ract ion was then d lgested wi th V ibr io cholera neuramimdare, which resul ted in the release Of d lsdcch l r ide and H - a c e t y l g a l a c t a ~ ~ t ~ ~ . 98).

D l l i a l lated OliqOIaCCharides - 1562-8 f r a c t i o n r h m e d I s ing le symet l i ca l peak On 8io-Gel P-2 g e l f i T t r a t T 0 n [ d o t t e d I?oc i o KS62-8 Of Flg. 5 ) and produced a d l l l i c h a r l d e a f t e r d e r i a l y -

m y a s described above. A f t e r m e t h y l a t i o n . i n t a c t o l i g o s a ~ c h a r i d e K562-BIV produced l a t i o n ( F i g . 5. K562-8). Th16 d l ldcchar ide was e r t a b l l r h e d to be GalE1*3GalNAcOH ~n the same

t r i - O m e t h y l N-nethylgalactosalninitol which can! r a t e d w i t h 1,4.5-tii-O-~thyig~lllrforaminltol pmduced from fe tu in o l igosacchar ide (F ig . BD?. 1562-8 wdl converted to monor ia ly la ted a l i go racchande a f te r treatment w i t h 02-3 s p e c i f i c n e w m i n i d a l e ( F i g . 9C) and t h i s m o n o r i a l y l a t e d ~ I i g o s a ~ ~ h a i i d e was converted to neutral d i s a c c h a r i d e w i t h V i b r i o iholera neuraminidase treatment (F ig , 90). There results are connstent w t h t h e f o l l o ~ s t r u c t u r e for K562-BIY.

NeuNAco2 \

NeuNAcZ-lGalL4GicNAc 0 6

NeuNAc2*3Gal * GalNAcOH a E \ e

(Structure I )

NeuNAcZ \ a

CL E E 6 3 NeuNAcZ*3Gal1*4GlcNAc - Gal * GalHAcOH

(St lYCtUm i l l

I n order to d i f fe ren t ia te these two p o s l i b l e s t r u c t u r e l . i n t a c t HL-60-8 ~ l i g ~ s d ~ c h d r i d e *a5

and borohydride-reduced HL-60-8 e l u t e d a t a p o s i t i o n where nonor ia ly la ted O l igosacchmde e lu ted subjected t o per iodate ox ida t ion and app l ied to a column of Bio-Gel P - 2 . The per lodate-ax id lzed

(Fig. IOA). Th is de r i va t i ve was then hydrolyzed wi th mi ld acld ("Smith Oegradat ion"1 and

where d l racchdr ide and trisaccharide e lu ted (F ig . 1OC). These results can be in te rpre ted I s subjected to Bio-Gel P-2 gel f i l t r a t i o n . The S r i t h degraded o l l go racchar ide e lu ted between

fo11m5. s ince c-6 o f g lucosamine conta ined t r i t ia ted hydrogen (dr ier i rks ind icate rad ioact ive hydrogen and rad ioact ive nanoracchar ider l .

C,-[*NeuNAc] - Gal

Gal - 0 - CH + Gal - 'GlCHAc - 0 - C - '1 I ,

n2c - on n (Oligo-1) (Oligo-2)

where C7-[MeuNlcl i s 5 - a c c t a ~ i d o - 3 . 5 - d i d ~ ~ ~ y - L - ~ ~ ~ b i " ~ - Z - h e p t " l ~ ~ ~ " i ~ a c i d .

A 1~411 peak e l u t e d a t 1 disacchar ide (Fig. IC€) war probab ly (~ l igo-1 s ince HL-60 c e l l s converted a m a l l anaunt o f r a d i o a c t i v i t y i n t o g a l a c t o s e (see Flg. IC). If t h e i n t a c t ollgorac- charide has a l i n e a r backbone II i n Structure 11, the f a l l ow ing pmduc t . wh ich i s larger than t r i sacchar ide . or Gal - 'GlcNAc Should be obtained.

CHZOH I I

I n2con

H - C - N H - A c

Gal - *GlcNlic - G a l - 0 - 1. - H

These result6 are c o n s i s t e n t w t h t h e f o l l o w n g st ructure ( S t r u C l U r e I) fop HL-60-BVI

NeuYco2*3GaIE1*4GlcNAcB1

'6 NeuNAc2*3Ga1$1-3GalNArDH

In order t o conf inn the above conclusion. the HL-60-8 o l l g o ~ a ~ ~ h a r i d e was d e r i a l y z e d w i t h m i l d a t i d h y d r o l y s i s and subjected to methy la t ion ana lys is . As r h w n i n Flg . BE. t h e d e r i a l y l a t e d HL-60-8 produced I.4.5-t~~-O-~ethyl-N-~thylgalactoraninitol and 3.6-dl-O-methyl-H~ethylglu- coramine a f t e r m e t h y l a t i o n . The results conf inned the above Conclusion.

Structural Variations of 0-Linked Oligosaccharides 12793

This conclusion was supported by the fact that neutral tr isaccharide I&% not formed frm K562 l e u l o r i a l i n w h l c h l l c t s branched 0-l inked ol igoraccharider. Instead. sldlyl 3-deoxy

was converted into 3-deoxy N-acetylqalactoraninital on ly a f te r V ib r io cholera neuraminidase N-acetrlsalact~raminitol was famed (Fractvmr 69-72 l n Flq. 5 . 1562-E). The r i a l y l a t e d fo rm

treatment (Fig. 91 and 8). The p'esmptive a - d e o x y ~ a l ~ c t o r a l ~ d l a t e r than

migrated a t the same posi t ion of the monor&charide released frm HL-BO-C trisdcchaTides (Tip. I-acetylqalactaraninrtol i n Bio-Gel P-2 gel filtration (Fractions 69-72 i n K562-C o f Fig. 5 ) and

6C and 0). Bared on these resul ts. the fo l lowing pepl ing react ion Can be proposed for X562 leukosial?n.

NeuNAcaZ b IeUNACG2 NaBH4/NaOH

NeuNA~dJGa101*3GalNAc*R \

(3-deoxylGalNA.cOH

3H-galacto~e labeled leukosialm frm HL-60 c e l l s * d l also subjected to al td l ine borohydr ide treatment. The OliqOf~cc.harides e lu ted in to three peaks by Scphadex G-50 gel f i l t r a t i o n , and these aliqaraccharider were estmated t o be NeuNAc .Gal . GlCNAc - GalHAcOH(1). NeuNAc. Gal- GlcIAc(3-dcoxy).GalNAccul(ll), and NeuIAc .Gal. GaldcOH ? I l l ) based on QAE-Sephadex column chrmtoqraphy and Bio-Gel P-2 ge l f i l t r a t i on be fo re and a f t e r neuraminidase treatment (data not shmn). There lerultl c o n f i m d t h e above conclurionr.

V O

t t t t t

I n K562 I r-"--i

L 30 40 50 60 7 0

FRACTION NUMBER

I K562 I K562 II

' i"

" 0

t t

h

HSB-2 II

t

30 40 50 60 7 0 30 40 50 60 7 0 FRACTION NUMBER

i T I 1 1 i 50

Fia. 4. QAE-Sephadex column ChraMtography of 3H-glucaramine labeled oliasraccharrder.

~ l i g o l a c c h d r i d e l 8 and C w e n rubJected t o QAE-Sephadex m l m n Chrmtography a s d e t a i l e d i n

to fur the7 analyr i l . No r a d i o a c t i v i t y was detected i n the fraCtion eluted with 200 M phosphate Experimental PlaCedYrel.'' The fractions indicated by hor1mntdl bars "eve p o l e d and subjected

buf fer . pH 7.4.

A

10 -

5 - n

B . GlcNAc f ract ion o f Fiq: 70. C. Fractions 68-73 of Fig. 110 ( ind icated by the bar] . D. Fractions 69-72 of K562-C friction i n r i g . 5.

12 794 Structural Variations of 0-Linked Oligosaccharides

N

i i i

A t GalNAcOH

'i

GlcNk

GBINAcOH t

O L , i 40 50 60 70 80

Fraction Number

F i l . 7: &io-Gel P-2 g e l f i l t r a t i o n o f o l i g o r a c c h a r i d e r a f t e r v a r i o u s e m - g l y c o s i d a s e t rea tmen ts .

A, K562-02 a f t e r t l e l t m e n t w t h E I C h e l l C h i a c o l i 0 - g a l a c t o s i d a s e . 0. N e u t r a l t e t r a s a c c h a r i d e (HSB-2-84) Of H S 0 m ( F i g . 5, HSB-2-01 a f te l t vea tmen t o f j ack bean

C. N e u t r a l t e t r a s a c c h a r i d e (HL-60-04) O f Hl-60 (see F i g . 5 , HL-60-81 a f t e l t r e a t m e n t Of jack

0. N e u t r a l t e t r a s a c c h a r i d e of HL-60 [ F i g . 5 . HL-60-81 a f t e r t r e a t m e n t d t h j ack bean

E . E E - g a l a c t o r i d a l e d i g e s t O f f r a c t i o n s 59-63 o f F i g . 70 [ i n d i c a t e d b y t h e bar) .

E - g a l l c t o l i d a l e .

bean E - g d l a ~ t o s i d a l e .

8 - g a l a c t o r i d a r e a n d 0-N-acetylglucoraninidase.

2, 3, a n d 4 i n d i c a t e t h e e l u t i o n p o s i t i o n s Of GaIE1-3GalNAcOH, GlcHlcE1~(GalE1*3)GalNAcOH. and Gal81~G1cNArE1*6(Gal81*3)GalNAcOH. r e r p e c t i v e l y .

d c t J a I I l l

2 4 6 8 10 12 14 16

cm from the Or~gm

Fig. E: Th in l aye r ch romatog raphy o f me thy la ted ga lac to ramin l to l r and l lUCoSamine ob ta lned f rom 1 Wethylated monolacchar ides were I vbJec ted t o t h in - l aye r ch romatog raphy . and 0.5 crn segments were s c r a p e d o f f t h e p l a t e and u s e d f o r l i q u l d s c i n t i l l a t i o n c o u n t i n g . The 11rou1 a . b , c and d i n d i c a t e t h e m i g r a t i o n p o s i t i o n l O f 1.4.5,6-tetra-O-methyl N-methylgll.ctol~.inltol, 1.3.4.5-

d i - 0 - m e t h y l N - m e t h y l g l u c o s m i n e . r e s p e c t i v e l y . t e t ra -0 -me thy l N-.ethylg l l .~ t~ l~ . in i to l . 1,4.5-tr i-O-methyl N-.ethylgdldctol..lnitol and 3.6-

A . Methy lated monoracchar ider frm K562-02.

8 . Methylated monosaccharides frm K562-CIII.

C. Methy lated monolacchar ides frm K 5 6 2 - C I I . K56Z-CIl S t i l l con ta ined a sma l l amount O f K 5 6 2 - C I I I b e f o r e m e t h y l a t i o n .

0. Methy lated rnonoracchar lder frm 1562-0.

E . Methy lated monolacchar ides fran d e r l a l y z e d HL-60.8.

F. Methy lated monosacchar ides f rom Hl-60-CIII ( F r a c t i o n s 43-46 ~n F i g . 5. HL-60-C were rech rmotog raphed On t h e same column and s u b j e c t e d t o m e t h y l a t i o n a n a l y s l l . )

4

2

0

z

E 4

5 2

' 1

X

€ 0

s >

0 U m o LT

1

0 2

1

0

A " i l i i

A n

-+/ 3b : A L 40 50 60 70

Fraction Numbel Fig. 9: 0io-Gel P-2 gel f i l t r a t i o n Of 1562 and HSB-2 o l i g o r a c c h a r i d e a f t e r n e u r a m i n i d a s e M. A. 02-3 spec i f i c neuramin idase t rea twent Of K562-C o l i g o l a c c h a r i d e .

8. Vib r io cho le ra neu ramin idase t rea twen t O f K562-C OllgOSaCChalides which were r e l i s t a n t t o ~ p e ~ n e u r a m i n i d a s e ( f r a c t i o n s 41-45 i n F i g . 9A).

C . 02.3 Spec i f i c neu ramin idase t rea tmen t o f K562-0 o l i go racchar ide .

0. V i b r i o c h o l e r a neuraminidase t r e a t m e n t o f K562-8 o l i gosacchar ides wh ich were r e l l I t a n t t o m e u r a . i n i d a r e t r e a t w e n t ( f r a c t i o n s 40-44 Of F i g . 9 0 .

E . 02.3 spec i f i c neu ramin idase t rea tmen t o f HSB-2-C o l i gosacchar ide .

1 2 3 and 4 i nd i ca te t he e lu t i on pos i t i ons o f GdlNAcOH, GdlE1-3GalNAcOH. GalBI.4GlcNAcE1.6 (~-d&)GalNACOH. and Ga101.4G1cNAcE1*6(GalE1*316alNAcOH, r e s p e c t i v e l y .

l A

Structural Variations of 0-Linked Oligosaccharides

A 3' 2' GIcNAc

I I I

-+/ 40 50 * 60 i o do

Fraction Number

A. 0-n-acetylglucor~ninidase digest of the trisaccharide (HSB-Z-C31 Trm HSB-2-C fraction (fractions 5 3 - 5 1 of HS8-2-C in Fig. 51. It eluted at the same Position as before digestion

12795