Enzymatic Basis for the Structural Changes of Asparagine-linked. Sugar Chains of Membrane Glycoproteins of Baby Hamster Kidney. Cells Induced by Polyoma ...
Vol. 260,, No. I, Issue of April
”HE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1985 by The AmericanSociety of Biological Chemists, Inc.
pp. 3963-3969 1985 Printed in &S.A.
Enzymatic Basis for the Structural Changes of Asparagine-linked Sugar Chains of Membrane Glycoproteins of Baby Hamster Kidney Cells Inducedby Polyoma Transformation* (Received for publication, May29,1984)
Katsuko Yamashita, Yoko Tachibana, Takashi Ohkura, and Akira KobataS From the Department of Biochemistry, Kobe University School of Medicine, Chuo-ku, Kobe, Japan and the SDepartment of Biochemistry, the Institute of Medical Science, the University of Tokyo, Shirokunedai, Mimto-ku, Tokyo 108, Japan
Previous studies indicated that enrichment of the addition to those derived from solidtumors. By studying the GlcNAc~l-AManal-, group with concomitantdebehavior of the glycopeptides obtained from baby hamster crease of the GlcNAc~l-4Manal+ group occurs in kidney (BHK’) cells and their polyoma transformants (Pythe complex-type asparagine-linked sugar chains of BHK cells) on a concanavalin A-Sepharose column, we sugthe membrane glycoproteins of baby hamster kidney gested that the increase in size of the sugar chains of glycocells transformed by polyoma virus. The enzymatic proteins in these transformed cells is probably inducedby an basis of the chemical change is reported inthis paper. increased number of outer chain moieties of complex-type By using oligosaccharides isolated from the urine of asparagine-linked sugar chains (2). Supporting evidence for patients with a variety of exoglycosidasedeficiencies, this assumption has been reported fromseveralresearch B-N-acetylglucosaminyltransferases in thecell homog- groups including ourselves(3-5). enate were successfully assayed separately. Both baby The recent establishment of controlled hydrazinolysis (6), hamster kidney cells and their polyoma transformants contain B-N-acetylglucosaminyltransferasesI, 11, IV, a chemical method forthe release of asparagine-linked sugar V, and VI, but not 8-N-acetylglucosaminyltransferase chains of glycoproteins,has enabled us to analyze quantitative III. The B-N-acetylglucosamine residue added by each and qualitative differences in plasma membrane glycoproteins B-N-acetylglucosaminyltransferase (GnT)is shown be- of cells (7,8) as well as purified glycoproteins. Comparative studies of the asparagine-linked sugar chains of membrane low. glycoproteins of BHK and Py-BHK cells using this method CnT ______ ClcNAc6% ClCNAC61 ------ CnT Ill 1 OFUC? have revealed that the transformation induces specific in2Mana 6 creases of the outer chains linked at the C-6 position of the CnT I 1 ------ CICNACBY‘ M a n d 4 residue of the trimannosyl cores of the complexCnT I V ------ ClcNAc6+ aM~n61~~lCNAC61~KilCN:C-A~n ,Manad3 type asparagine-linked sugar chains (9). The basis for this CnT I ___--ClcNAc61/L phenotypic change couldarise from an altered B-N-acetylgluCnT V I ---- 6-N-Acetylglucosaminyltransferare responsible for the cosaminyltransferaseactivity originating from altered genetic formation o f cal61+K;lcNAc61+3 repeating unit. control. However, it could also arise fromchangesin the Comparative studies of the specific activities of the membrane components since such transferases are embedded five 8-N-acetylglucosaminyltransferasesinthe two in the Golgi membrane. In order to elucidate which of these cell lines revealed that the value of B-N-acetylglucos- possibilities is correct, we have analyzed the activities of a aminyltransferaseV in the polyoma transformantwas series of B-N-acetylglucosaminyltransferasesresponsible for twice of that in the normal cells, while those of the the formation of the outer chain moieties of complex-type other four transferases in thetwo cell lines were not sugar chains in BHK cellsand Py-BHK cells. significantly different. Therefore the increasein 8-Nacetylglucosaminyltransferase V ma> bethedirect EXPERIMENTAL PROCEDURES~ cause of the changes found in the sugar chains of surface glycoproteins in baby hamster kidney cells transRESULTS AND DISCUSSION formed by polyoma virus. Thus far, six j3-N-acetylglucosaminyltransferases,shown in Fig. 1as GnT I to VI, are considered to be responsible forthe +
Several differences have been found in the structures of sugar chains of glycoproteins producedin normal and malignant cells. One of them is a characteristic increase in the molecular weightof the asparagine-linked sugar chains which has been widely observed in the plasma membrane glycoproteins of transformed and malignant cells from a variety of mammals (1);malignant cells in culture were examined in
The abbreviations used are: BHK, baby hamster kidney; XylNAc, N-acetylxylosaminitol.SubscriptOTisused to indicateNaBaH,reduced oligosaccharides. In the same way, subscript OH is used to indicateNaBH,-reducedoligosaccharides.Allsugarsmentionedin this paper were of the D-configuration except for fucose which has the L-configuration. Portions ofthis paper (including “Experimental Procedures” and Tables 1-111) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid ofa standard magnifying glass. * This work was supported by a Grant-in-Aid for Special Project Full sizephotocopiesareavailablefromthe Journal of Biological Research, Cancer-Bioscience, from the Ministry of Education, Sci- Chemistry, 9650hckville Pike, Bethesda, MD 20814. Request Docence and Culture of Japan. The costa of publication of this article ument No. “-1621, cite the authors, and include a check or money were defrayed in part by the payment of page charges. This article order for $2.40 perset of photocopies. Full size photocopies are also must thereforebe hereby marked“advertisement” in accordance with included in the microfilm edition of the Journal that is available from 18 U.S.C.Section 1734 solely to indicate this fact. Waverly Press.
3963
GlcNAc Transferases in Transformed Cell
3964
CnT V I
----
6-N-Acetylglucosaminyltransferase responsible for the formation of CaI61+4cIcN~c61+3 repeating unit.
FIG. 1. A structure showing the possible 8-N-acetylglucosamine residues that can be attached to the trimannosyl cores of the complex-type asparagine-linked sugar chains. fl-N-Acetylglucosaminyltransferases (abbreviated in this paper as GnT) re-
D
sponsible for the addition of each P-N-acetylglucosamine residue will be called by the number indicated in thisfigure.
E addition of different 0-N-acetylglucosamine residues to the trimannosyl core. GnT VI catalyzes the addition of the 0-Nacetylglucosamine residue which is not linked directly to the core but to the galactosyl residue of the Galo14GlcNAc outer chainsto initiate formation of the N-acetyllactosamine repeating structures. Since the purpose of this study was to determine the concentrations of all six 0-N-acetylglucosaminyltransferases, we had to devise special means for the accurate determination of the activity of each transferase in cell homogenates. Like many other glycosyltransferases, the six 0-N-acetylglucosaminyltransferaseshave been shown to have fairly strict specificities toward the acceptor sugar chains: GnT I (10, l l ) , I1 (ll),I11 (12), IV (13), V (14), and VI (15). Therefore, it was considered possible that theactivity of each enzyme might be determined ina cell homogenate, if an appropriate sugar chain was chosen as anacceptor. So we have thoroughly investigated the radioactive products obtained by incubation of the oligosaccharides listed in Table I with UDP-N-a~etyl[U-'~C]gluco~arnine and homogenates of BHK cells or of Py-BHK cells as sources of enzymes. Except for Mz.GN F GN, GN .Mz.GN, and GN4.MS.GN, all oligosaccharides listed in Table I can be converted to either one or two radioactive oligosaccharides by such incubations. This evidence indicated that GnT I11 might be deficient in both BHK cells and Py-BHK cells, because GN,. M,-GN served as a good acceptor for this enzyme in homogenates of hen o v i d ~ c tThis . ~ inference was further confirmed by the complete absence of the bisecting N-acetylglucosamine residue in all radioactive products described below. Absence of the enzyme in BHK cells and in Py-BHK cells is also reflected in the structures of asparagine-linked sugar chains of glycoproteins from both cells, which completely lack the bisecting N acetylglucosamine residue (9). Structural Analyses of the Radioactive Products from Mz. GN, M3.GN, and Ms. GN-Only one radioactive product was obtained from Mz.GN and Ms. GN (Fig. 2, A and B, respectively). From the differences in mobilities between acceptors and products in the Bio-Gel P-4 column, it was concluded that one N-acetyl[U-'4CC]glucosamineresidue was added to each oligosaccharide acceptor. When these radioactive products were reduced with NaBH4 andthen incubated with diplococcal fi-N-acetylhexosaminidase,4all the radioactivity +
K. Yamashita, T. Ohkura, and A. Kobata, unpublished result. diplococcal 0-N-acetylhexosaminidase cannot cleave the GlcNAcpl+4Man linkage in GlcNAcfll+SManal+ 6[GlcNAc~l+4(GlcNAc~l+2)Manal+3]Man~1+4GlcNAc~~ as reported previously (16), the enzyme readily cleaves the linkage in
'Although
GlcNAc~1+2Manal~6[GlcNAc~l+4(GlcNAc~l+2)Manal+3] Manpl4GlcNAc. Therefore, the oligosaccharide must be reduced beforehand in order to use the substrate specificity of diplococcal 0N-acetylhexosaminidase for the linkage determination of 0-N-acetylglucosaminyl residues in the outer chain moiety.
A
A \ G
285
255
225 ELUTION VOLUME (ml)
b
5
FIG.2. Bio-Gel P-4 column chromatography of the radioactive products from Ma*GN, Ma*GN,and Ms-GN and their enzymatic and chemical degradation products. Only data obtained from experiments where BHK cell homogenates were used as sources of enzymes are presented; the same results were also found in studies with Py-BHK cell homogenates. The block arrows a t the top of the figure indicate the elution positions of glucose oligomers (numbers indicate the glucose units), and the white arrows indicate those of authentic oligosaccharides as follows: I , Manal+BManfll+ 4GlcNAcm; 2, Manfl14XylNAcW; 3, N-acetylglucosamine. A , B, and C, the radioactive products obtained from Mz. GN, Mg. GN, and MI. GN, respectively (the block triangles indicate the elution positions of acceptor oligosaccharides);D,the radioactive peaks in A , B, and C reduced with NaBH, and then incubated with diplococcal 0-N-acetylhexosaminidase; E, the radioactive peak b in C incubated with jack bean a-mannosidase; F, the radioactive peak in E reduced with NaB'H, and incubated with diplococcal P-N-acetylhexosaminidase (dotted line, radioactivities determined on an aliquot of each fraction by liquid scintillation spectrometer; solid line, obtained by monitoring effluent with a radioanalyzer); and G,the dotted line (tritium labeled) peak in F subjected to periodate oxidation.
was recovered in a peak which corresponded to N-acetylglucosamine (Fig. 2 0 ) . This result indicated that all radioactive N-acetylglucosamine residues added to the two acceptor oligosaccharides occur as the GlcNAcfil~ZMan group because 8-N-acetylhexosaminidasecan only cleavethis linkage among the five 0-N-acetylglucosaminyl linkages connected directly to thetrimannosyl core shown in Fig. 1 (16). Two radioactive products were obtained from M s . GN (Fig. 2C). From their mobilities on the Bio-Gel P-4 column, it was concluded that peaks a and b contain two and one radioactive N-acetylglucosamine residues, respectively. All radioactivity in both peaks after NaBH4reduction was also released as free N-acetylglucosamine by diplococcal 8-N-acetylhexosaminidase treatment, which showed that all the N-acetylglucosamine residues added occur as theGlcNAcpl-2Man group. No degradation was observed when the radioactive peak in Fig. 2A and the radioactive peak a in Fig. 2C were incubated with jack bean a-mannosidase (2 units/ml) (data not shown). These resultsindicated that theradioactive product from MZ. GN and the larger radioactive product from M3.GN should have the structure shown in Table 11. The radioactive peak in Fig. 2B was converted to a radio-
GlcNAc Transferasesin Transformed Cell active oligosaccharide with the same mobility as the radioactive peak in Fig. 2A by the a-mannosidase digestion (data not shown). This result showed that thetwo nonreducing terminal a-mannosyl residues of the Manal-&(Manal-d)Mana14 group were not substitutedby the newly added 8-N-acetylglucosamine residue. Therefore, the structure of the radioactive product from M,. GN should be that shown in Table 11. When the radioactive peak b in Fig. 2C was incubated with jack bean a-mannosidase, one mannose was removed (Fig. 2E).Inorder to determine which of the two a-mannosyl residues was removed byenzymatic digestion, the radioactive component in Fig. 2E was reduced with NaB3H4 and then digested with diplococcal /3-N-acetylhexosaminidase.As shown in Fig. 2F,two radioactive products were obtained by this treatment. Thepeak with the same mobility as N-acetylglucosamine contained only '"C radioactivity, while the peak with a mobility of 4.2 glucose units contained only 3H radioactivity. Therefore, this peak should correspond to Manal3 and/or 6Man/31+4GlcNAc~.When the radioactive trisaccharide was subjected to periodate oxidation, all the radioactive component was converted to Man/314XylNAcoT (Fig. 2G).This result indicated that the radioactive trisaccharide in Fig. 2F should be Manal+3Man/314GlcNAc~ andnot Mana14Man/31+4GlcNAcoT, because the latter trisaccharide is converted to N-acetylxylosaminitol by periodate oxidation (22). Based on the analytical results, the radioactive peak b in Fig. 2C was presumed to have the structure shown in Table 11. The experimental resultsso far described indicated that all three oligosaccharides can be acceptors of only GnT I. In the case of M3.GN, however, the product acts as agood acceptor of GnT 11, producing a di-N-acetylglucosaminyl derivative. This result is in good agreement with the reports of Harpaz and Schachter (11) and of Oppenheimer et al. (17) but is different from the report of Mendicino et al. (18) which indicated that M3-GN2.Asn canbe an acceptor of both GnT I and GnT 11. Therefore, the activity of GnT I1 alone could be determined by using GN. M3.GN. When the pentasaccharide was used as an acceptor, a single radioactive product corresponding to peak a in Fig. 2C was detected (data not shown). Structures of the Radioactive Products from GN2.MS.GN and GN3. M3-GN-Although a single radioactive peak was obtained from GN2. M3-GN when BHK and Py-BHK cell homogenates were used as sources of enzyme, the elution patterns of the two reaction products were slightly different (Fig. 3,A and B, respectively). The mobilities of the products on the Bio-Gel P-4 column indicated that anN-acetylglucosamine residue was added to the hexasaccharide acceptor. All the radioactive N-acetylglucosamine residues should have the @-anomeric configuration because all radioactivities in the peaks were released as N-acetylglucosamine by incubation with jack bean @-N-acetylhexosaminidase(data not shown). When the radioactive peaks in Fig. 3,A and B, were reduced with NaBH, and subjected to paper chromatography, both of them were separated into two radioactive components (Fig. 4, A and B ) . The mobilities of the two radioactive components a and b were the same asauthentic GlcNAc@l&(GlcNAc@l+2)Manal+6(GlcNAc~l+2Mana1+3)Man,91+ 4 G l c N . 4 ~ 0and ~ GlcNAc@1+4(GlcNAc@l+2)Manal+ 3(GlcNAc@1+2Mana14)Man@14GlcNAcoT, respectively (Fig. 4, A and B ) . Incubation with diplococcal @-N-acetylhexosaminidase released one nonradioactive N-acetylglucosa-
3965
I ' 190
210
230
250
ELUTION VOLUME (ml)
FIG.3. Bio-Gel P-4 column chromatography of the radioactive products from GNa*Ms.GN and GNa-Ms.CN and their enzymatic degradation products. The block arrows are the same as in Fig. 2. The white arrows indicate the elution positions of authentic oligosaccharides: I , GlcNAc@1~(GlcNAc@l+2)Manal+ G(Manal+3)Man@1+4GlcNAc0~; 2, GlcNAcj31+4Manal+ 3(Manal+6)Manj314GlcNAco~.A and B, the radioactive products obtained from GNz.Ma. GN by using BHK cell homogenates and PyBHK cell homogenates as enzyme sources, respectively; C, the radioactive peak a in Fig. 4A incubated with diplococcal @-N-acetylhexosaminidase; D, the radioactive peak b in Fig. 4A incubated with diplococcal @-N-acetylhexosaminidase;E, the radioactive products obtained from GN3. Ma.GN by using either a BHK cell homogenate or Py-BHK cell homogenate as anenzyme source; F, the radioactive peak in E, reduced with NaBH, and then incubated with diplococcal 8-N-acetylhexosaminidase.The black triangles in A and B indicate the elution positions of GNZ. M3. GN and that in E the position of GN3. M3. GN.
N
' 0 4
I
2
.L
L
DISTANCE FROM ORIGIN (cm) FIG. 4. Paper chromatographic separation of the two isomeric radioactive products obtained from GNa*Ms'GN. Arrows indicate the positions where authentic oligosaccharides migrated as follows: I , GlcNAc@l4(GlcNAcj3l+2)Mana1+6(GlcNAc~1+ 2Mana1+3)Manj31+4GlcNAc~; 2, GN3.M3.GN.A and B, the radioactive peaks in Fig. 3, A and B were reduced with NaBH, and then subjected to paper chromatography using ethyl acetate:pyridine:acetic acidwater (5:5:1:3) as a solvent for 48 h, respectively.
mine from component a and two nonradioactive N-acetylglucosamines from component b of the BHK product (Fig. 3, C and D, respectively) and of the Py-BHK product (data not shown). As reported in the previous paper (16),the enzyme converts GlcNAcpl4(GlcNAc~l~2)Manal4(GlcNAc@l+2Manal+3)Man@l+4GlcNAco~ to GlcNAc@l+ 6(GlcNAc@l+2)Mana14(Manal-*3)Man@1+4GlcNAcoT and GlcNAc@1-*4(GlcNAc@1+2)Manal~3(GlcNAc~l+
GlcNAc Transferases in Transformed Cell
3966
2Manal-*G)Man@1+4GlcNAco~to GlcNAc@1+4Manal+ diplococcal @-galactosidase,one, two, and three galactoses 3(Mana14)Manj31+4GlcNAc~.Therefore, the structures were removedfrom these radioactive products (solid line peaks of components a and b should be [“C]GlcNAc@1+6(Glc- in Fig. 5, B, D, and F, respectively). All radioactivity in the three peaks was completely converted to a disaccharide with NAc@1+2)Manal+6(G1cNAc@l~2Manal+3)Man@l+ 4GlcNAcoH and [“C]GlcNAc@1+4(GlcNAc@1~2)Mana1+ the same mobility as authentic GlcNAc@1+3Galby endo-@3(GlcNAc@1+2Mana14)Man@1+4GlcNAcoH which were galactosidase digestion (dotted line peaks in Fig. 5 , B , D, and formed by the action of GnT V and IV, respectively. There- F). That thedotted l i n e peaks in Fig. 5, B , D, and F have the fore, the relative activities of GnT V and IV can be determined structure [“C]GlcNAc@14alwas confirmed by the followby measuring the radioactivities in components a and b. In ing experiments. After reduction with NaB3Hr,the radioactive contrast, inthe case of GN3.M3. GN,the same elution profile oligosaccharides were digested with diplococcal @-N-acetylfor the radioactive product was obtained by the use of GN3. hexosaminidase. All of them were completely converted to a M3.GN as acceptor and BHK homogenate (Fig. 3E) or Py- mixture of [“C] N-acetylglucosamine and [3H]galactitol, BHK homogenate (data not shown) as the enzyme source. which were identified by paper chromatography using buThe mobilities of the radioactive products indicated that an tanol-1:ethanol:water (4l:l by volume)as a solvent and paper N-acetylglucosamine residue was added to the heptasaccha- electrophoresis with borate buffer (19). Although the linkage ride. All the radioactivity contained in the products was between the N-acetylglucosamine and the galactose of the released as N-acetylglucosamine by incubation with jack bean disaccharide fragment was not confirmed, the structures of @-N-acetylhexosaminidase(data notshown). When incubated the asparagine-linked sugar chains of the surface glycoprowith diplococcal @-N-acetylhexosaminidase,all radioactivity teins of BHK and Py-BHK cells (9) and the substrate speciremained in the oligosaccharide whose size was decreased by ficity of the endo-19-galactosidase (26) suggested that the 1.0 glucose unit indicating that anN-acetylglucosamine resi- radioactive 8-N-acetylglucosamine residues were added at the due wasremoved by the enzyme digestion (Fig. 3F). This C-3 position of one of the terminal @-galactosylresidues of result showed that only [‘4C]GlcNAc~1+6(GlcNAc~l~2) the threeoligosaccharide acceptors by the action of GnT VI. Manal+6[GlcNAc~1+4(GlcNAc@l+2)Manal+3]ManOptimal Conditions for the Assay of Each j3-N-Acetylglucos/314GlcNAc was produced from GN3 Ms. GN by the action aminyltranaferase-The results described so far indicated that of GnT V. the concentrations of GnT I, 11, IV, V, and VI in BHK cells Structures of the Radioactive Products from G2.GN2.M3. and inPy-BHK cells can be determined by using appropriate GN, GB GN3.M 3 .GN, and G4.GN4.M 3 .GN-Only one N-ace- oligosaccharides listed in Table I. Furthermore, important tylglucosamine residue was added to the three oligosaccharide evidence concerning the substrate specificities of GnTs has acceptors: G2. GN2. Ms. GN (Fig. 5 A ) , GB.GN3.M3. GN (Fig. been revealed by this study. That the N-acetylglucosamine 5C), andG4. GN4.M3.GN (Fig. 5E). By incubation with residue cannot be transferred to Mz.GN .F .GN indicated that GnT I1 in BHK andPy-BHK cells cannot use a linear sugar chain, unless a fucose residue inhibits GnT 11. That GN M2. GN cannot be an acceptor of any @-N-acetylglucosaminyltransferases in these cells indicated that GnTIV and GnTV cannot use GlcNAc~1+2Mana1+3Man@1+4GlcNAc and GlcNAc~l~2Manal-X1Man@l+4GlcNAc as theiracceptors, respectively. Therefore, the oligosaccharide substrate of these transferases must containa trimannosyl core. Optimal conditions for the assay of each enzyme were studied in more detail. All five 0-N-acetylglucosaminyltransferases showed rather broad pH optima ranging from pH 6.0 to 7.0. Therefore, pH 7.0 was used for the assay of all enzymes to suppress the action of lysosomal glycosidases as much as possible. GnT V did not need added Mn2+ as reported by Cummings et al. (14), but all other enzymes required this cation. Therefore, this cation was added in a final concentration of 20 mM to all reaction mixtures, except for GN2.Ms. GN which was incubated with and without MnZ+to determine the activity of GnT IV GnT V and GnTV only. Preliminary experiments revealed that the amounts of radioactive N-acetylgIucosaminetransferred to oligosaccharides affer 16-h incubation were the same when the concentration ELUTION VOLUME (mll of UDP-GlcNAc was higher than 2.5 mM. We also found that FIG. 5. Bio-Gel P-4 column chromatography of the radioalmost no donor nucleotide remained after 16-h incubation active products from Gs*GNs.Ma.GN, Gs*GNs*Ma*GN, and when used in a concentration of 0.5 mM. Therefore, we chose G4*GN4*&*GNand their enzymatic degradation products. to use UDP-GlcNAc (2.5 mM), so that sufficient amounts of The block arrows are the same as in Fig. 2, and the white arrow indicates the elution position of GlcNAcgl4Gal. A, C, and E, the the donor remained when the reaction was over. The kinetic properties of each 8-N-acetylglucosaminylradioactive producta obtained from G2.GN2.Ms.GN, Gs.GNa.Ms. GN, respectively (the block triangles indicate transferase were studied by using oligosaccharides in Table I GN, and G4.GN4.Ms. the elution positions of acceptor oligosaccharides); B, D,and F, the as substrates. Although only the dataobtained by using GN2 radioactive peaks in A, C, and E incubated with diplococcal 8Ms. GN are shown in Fig. 6,the reaction galactosidase (solid line) and then with endo-b-galactosidase (dotted Ms. GN and Gz. GNz. rates were proportional to protein concentration and were l i n e ) , respectively.
-
A ’
A’
h
+
GlcNAc Transferasesin TransformedCell
i3t
Y
3967
A t FIG. 7. Pathwayshowingthesynthesis of complextype sugar chains.M,Man; G, Gal; GN,GlcNAc, GT,galactosyltransferase.
FIG.6. Kinetic data of GnT IV, V, and VI.A and C,effect of incubation time on the reaction rate determined with BHK or PyBHK cell homogenate (150 pg of protein), respectively. The conditions of incubation are the same as described under "Experimental Procedures" except that the time of incubation was varied as indicated. B and D,effect of enzyme concentration on the reaction rate determined with BHK orPy-BHK cell homogenate, respectively. The conditions of incubation are the same as described under "Experimental Procedures" except that protein concentration was varied as indicated. e-0 and U, the activities of GnT IV and V, respectively, as determined by using GNa-Ma.GN as an acceptor. The activity of each enzyme was determined after separating the products of the two enzymes by paper chromatography as described in the text. A-A, the activity of GnT VI as determined by using GI. GN, .Ms. GN as anacceptor.
linear for at least 4 h in all cases. Comparison of the Concentrations of Five N-Acetylglzzosaminyltransferases in BHK Cells and Py-BHK CeUs-The activities of the five B-N-acetylglucosaminyltransferasesin the homogenates of BHK and Py-BHK cells were determined using the optimal conditions described aboveand the results are summarized in Table 111. Becausepeak a in Fig.2C contains two N-acetyl[U-"C]glucosamine residues, the activity of GnT I was calculated on the basis of the radioactivity in peak b plus half of that in peak a in Fig.2C, and the activity of GnT I1 was estimated on the basis of half the radioactivity in peak a. The data indicated that GnT I and GnT I1 activities are one order higher than other 0-N-acetylglucosaminyltransferasesin both cell homogenates. As is evident from the Py-BHK/BHK ratio of each enzyme in the two homogenates,both cells havethe same concentrations of GnTs I, 11, IV, and VI. In contrast, the activity of GnT V is twice that in Py-BHK cells irrespective of the different oligosaccharide acceptors. The results indicated that the increase in concentration of GnT V may be the direct cause of the structural change in the asparagine-linked sugar chains of BHK cells by malignant transformation. In Fig. 7, a biosynthetic network responsible for the formation of a series of complex-type asparagine-
linked sugar chains is shown. Because of the acceleration of the two pathways indicated by the bold arrows, Py-BHK cells synthesize more tri- and tetra-antennary sugar chains with the GlcNAcj31-&Manal+ group. Van den Eijnden and Schiphorst (20) recently reported that GnT VI purifiedfrom Novikoff tumor cell ascites fluid works most favorably on GalB14GlcNAcp1
I 6
sugar chains with the
group. Man 2
If it
f Gal#?l4GlcNAc/31 is a general characteristic of GnT VI, incrementa of the highly branched complex-type asparagine-linked sugar chains with the Gal@l+IGlcNAcBl-&Man group may enhance the formation of sugar chains with N-acetyllactosamine repeating structures in their outer chain moiety. This may explain why tri- and tetraantennary complex-type sugar chains with Nacetyllactosamine repeating structures of the membrane bound glycoproteins of Py-BHK cells showed a higher turnover rate thanthose of BHK cellsas reported in theprevious paper (9). Ackrwwkdgments-We wish to express our gratitudeto Dr. Alistair G. C. Renwick for his critical reading of this paper and to Dr. Shunichi Nakamura for his technical assistance in performing this study. Thanks are also due to the skilled secretarial assistance of Yumiko Kimizuka. REFERENCES 1. Warren, L.,Buck, C.A., and Tuszynski, G. P. (1978)Biochim. Biophys. Acta 616,97-127 2. Ogata, S.,Muramatau, T., and Kobata, A. (1976)Nature 269, 580-582 3. Santer, U. V., and Glick, M. C. (1979)Bkhemistry 18,25332540 4. Takasaki, S., Ikehira, H., and Kobata, A. (1980)Biochern. Biophys. Res. Commun. 92,735-742
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to
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GlcNAc Transferases in Transformed Cell
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41.1, 42.6 (41.71
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20.5, 21.3 120.91
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3969
