[CANCER RESEARCH 40, 1550-1 557, May 1980]0008-5472/80/0040-OOOOS02.00
Surface Labeling of Sialic Acid Residues in BHK Cells and ViralTransformants1
Harold Flowers2 and Mary Catherine Click3
Department oÃPediatrics. University of Pennsylvania School of Medicine, and The Joseph Stokes, Jr. Research Institute, Children's Hospital of Philadelphia,
Philadelphia, Pennsylvania 19104
ABSTRACT
Conditions were found for the direct radioactive labeling ofglycoproteins on the membranes of baby hamster kidney fibro-
blasts and their Rous sarcoma virus transformants with negligible loss of viability. Sialic acid residues were specificallydetected by oxidation of the cells with periodate followed byreduction with sodium borotritide. Highly specific incorporationof radioactivity was demonstrated into surface membranesisolated as whole structures from these cells. Comparison wasmade with a known method which labeled galactose residuesutilizing galactose oxidase. It was suggested that, when surfacelabeling procedures are used which are not extremely gentleto the cells, cell fractionation should be performed.
Gel electrophoresis in sodium dodecyl sulfate enabled comparisons to be made of the sialic acid-containing glycoproteins
in the surface membranes from the transformed cells and theirnormal counterpart, and a number of conclusions were drawnon the exposure of different glycosylated macromolecules tothe external environment. These included: (a) differences wereobserved in the molecular weights and number of species ofthe prominent sialic acid-containing glycoproteins; (b) labeling
of isolated membranes showed additional glycoproteins aboveM.W. 150,000; (c) the method of oxidation prior to labelingyielded different externally oriented glycoproteins in the normalcells, although the transformants showed no difference; and(d) a number of other differences depended on the method ofcell harvest prior to labeling.
Transformed cells appeared to adhere more strongly to theplastic surface of the culture flasks and required longer treatments with a variety of reagents than did their normal counterparts to ensure their complete dislodgement.
INTRODUCTION
The external orientation of plasma membrane carbohydratesin various cells has been demonstrated by a number of methods(4, 11, 19, 33), and information has been obtained on membrane glycoproteins labeled by supposedly nonpenetrating reagents and accordingly depicted as externally oriented (2, 11,33). The use of these labeling techniques has also revealeddifferences between normal cells and their virally transformedcounterparts, although the full significance of these findings isnot yet clear. Of special interest has been the demonstration ofa slow-migrating band on gel electrophoresis in sodium dodecyl
sulfate, corresponding to a protein subunit with an apparentmolecular weight of 200,000 to 250,00 in a number of normal
' Supported by American Cancer Society, Grant BC 109, and NIH Grants CA
14037 and CA 14489.2 Permanent address: Department of Biophysics, Weizmann Institute of Sci
ence, Rehovot, Israel.3 To whom requests for reprints should be addressed.
Received May 9, 1979; accepted January 31. 1980.
cells, but which is reduced or disappears on transformation ofsome cells (6, 19, 21, 37). Fibronectin contains carbohydrate(19, 21, 42) and has been labeled by various external labelingtechniques; however, it is a matrix component rather than amembrane protein (5, 7, 18), suggesting a need for morespecific membrane markers. Additional differences of unknownsignificance have also been manifested on careful comparisonof electrophoretic patterns of labeled animal cells or membranes before and after viral transformation (1, 12, 17, 21).
An alternative approach to investigating differences in thecomposition and structure of normal and transformed cells hasbeen to study glycopeptides removed from the cell surface bymild proteolysis (trypsinization). It was shown that trypsinatesof virally transformed cells and of cells isolated from tumorsdiffered from those of their normal counterparts (12, 35, 37,39). After pronase digestion of the trypsinates, rapidly migrating material was observed on Sephadex G-50 columns in the
transformed and tumor cells which was greatly reduced in thenormal cells. Sequence analysis of the oligosaccharide moietyof one of these glycopeptides from the transformed cellsshowed a 3-branched structure with A/-acetylneuraminic acid
in the terminal position on each branch (16, 35).The prevalence of sialic acid-containing glycoproteins in the
surface membrane makes the labeling of these residues one ofthe more specific markers for the surface membrane (15). Infact, many cell types contain more than 70% of their total sialicacid content in the surface membrane (13). The direct labelingof the sialic acid residues on the surface membrane of cells inculture has not been reported previously, although others havelabeled erythrocyte stroma (27, 30) or lymphoid cells (8).
Among the difficulties in the previous procedures used forexternal labeling of cells have been the prolonged reactiontimes and somewhat harsh treatments of the various cells withthe reagents. The known fragility of a number of cell typesmakes interpretation of the data difficult, particularly whensmall numbers of lysed cells contribute large amounts of par-
ticulate protein. The rapid as well as gentle procedure described in this paper circumvents this difficulty, and only thesialic acid residues are labeled. The external position of thelabel was verified by isolation of whole surface membranes.The specificity for the surface was further shown by the markedconcentration of radioactivity in the cell membrane in contrastto other cell fractions. Using this procedure, a number ofdifferences in the sialic acid-containing glycoproteins after
virus transformation were seen which heretofore have not beenreported.
MATERIALS AND METHODS
Cell Culture
Baby hamster kidney fibroblasts, BHK2i/C13 and this clone
transformed by the Bryan strain of Rous sarcoma virus, CÃa/B4, were grown on 150-sq cm flasks (Falcon) to the upper logphase of growth in an atmosphere of C02. Eagle's minimal
essential medium (Flow Laboratories) supplemented with 5%fetal calf serum (Flow Laboratories) and 10% tryptose phosphate broth (Difco) was used as growth medium (3). In no casewere cells used beyond passage 15. Cells were inoculated at2.5 x 106/flask. When the cells were labeled metabolicallywith L-t14C]fucose, 12 juCi/flask were added 48 hr before
harvest in 25 ml of fresh medium.
Cell Harvest
After removal of the growth medium and 4 washings with asolution appropriate for each procedure as described below,the cells were removed from the monolayers by one of thefollowing 4 alternative techniques, using 3 ml of the appropriatereagents per flask.
Method 1: Incubation with Trypsin. Gentle bathing andshaking at room temperature of each culture flask with asolution of 0.18 mg of trypsin in 0.16 M NaCI led to completedislodgement of BHK21/d3 and d3/B4 cells within 15 and 20min, respectively. Proteolysis was stopped by the addition ofsoybean trypsin inhibitor in an amount necessary to neutralizethe trypsin. These cells were denoted as "trypsinized cells."
Method 2: Treatment with Disodium EDTA. The addition of0.2% disodium EDTA in TBS" followed by shaking and gentle
aspiration of the cells at room temperature led to their releasewithin 6 to 10 min from the monolayer in the case of BHK2i/C13 cells, although up to 1 hr was necessary for completerelease of the transformed cells.
Method 3: Treatment with Isotonic Phosphate. BHK2i/Ci3cells were loosened after 10 to 15 min with isotonic phosphate,pH 7.4, at room temperature and could be harvested by aspiration. Under the same conditions, the transformed cells remained attached as a monolayer.
Method 4: Treatment with PBS. After addition of PBS, pH7.0, to the monolayers, the flasks were kept for 5 min at 4°
and then returned to ambient temperature. Shaking accompanied by aspiration for 15 to 20 min led to complete release ofBHK21/C,3 cells, but again C,3/B4 cells remained attached tothe flasks.
Preparation of Cells for Labeling
Cell suspensions were centrifuged at 230 x g and washedonce with one of the following solutions, depending on themethod used for harvesting: (a) 0.16 M NaCI; (Ö)TBS containing 0.05 M NaCI2, then TBS; (c) isotonic phosphate, pH 7.4;and (d) PBS, pH 7.O. The cells were then washed twice withthe appropriate buffer to be used in the labeling reaction. Eachwashing procedure consisted of gentle mixing and centrifuga-
tion at 230 x g.
Radioactive Labeling
Carbohydrate residues of cells removed from the monolayersor isolated cell membranes were oxidized by either periodateor galactose oxidase and then labeled with tritium by reduction
4 The abbreviations used are: TBS, 0.02 M Tris-HCI, pH 7.5. in 0.15 M NaCI;
isotonic phosphate, 0.1 M sodium phosphate buffer, pH 7.4; PBS, 0.05 M sodiumphosphate buffer, pH 7.0:0.08 M NaCI; SDS. sodium dodecyl sulfate.
of the products with NaB3H4. In some cases, cells were labeled
directly on the monolayer.
Periodate:NaB3H4 Labeling
Various times and conditions of incubation with the reagentswere investigated, and the following were selected as affordinggood incorporation of radioactivity with high cell viability (90%or above). To a cell suspension in 0.5 ml of isotonic phosphate,pH 7.4, containing 0.5 to 1.0 x 108 cells, were added 10 /il of
0.1 M NalO4 solution, and the mixture was kept for 10 min atroom temperature with occasional gentle mixing. Cold buffer(6 to 8 ml) was added, the suspension was centrifuged and thepellet was washed twice more with buffer. A solution (1 to 2/il) of NaB3H4 containing 500 /¿Ciin 0.01 N NaOH was added tothe pellet at 5°,and the mixture was kept at 5°for 5 min with
occasional gentle mixing. The cells were then washed 3 timeswith buffer and twice with 0.16 M NaCI. Cells which were usedas controls underwent the same treatment, except the periodate step was omitted. Aliquots of the final cell suspensionswere collected for protein and radioactivity determinations andcell count, including viability assay by uptake of trypan blue asmeasured in a Biophysics Cytograf. The radioactivity of thesupernatant solutions was examined as a check on the efficiency of the washing procedure.
To label the cells directly on the monolayer, the medium wasremoved and, after being washed with the appropriate buffer,the cells were treated directly with NalO4 in 2 ml of eitherisotonic phosphate, pH 7.4, or PBS, pH 7.O. In the first case,cells began to dislodge during treatment and were collectedand washed in centrifuge tubes. The pellets were then tritiatedas described. With careful handling, most BHK2,/Ci3 cells andall Ci3/B4 cells remained attached to the flasks in PBS, pH 7.0,throughout the complete cycle of NalO4-NaB3H4 treatment.
They were then harvested by trypsinization as described previously, and the trypsinates were dialyzed thoroughly againstwater at 4°, lyophilized, and frozen at —¿�40°until further
examination. Subsequent treatments of the trypsinates whichincluded digestion with pronase and chromatography on Seph-adex G-50 columns have been described (3).
Galactose Oxidase:NaB3H4 Labeling
Cells (1 x 108) in PBS, pH 6.0, were incubated with Vibrio
cholerae neuraminidase (25 units to 0.5 ml of cell suspensions).After 15 min at 37°,the cells were washed twice with PBS, pH
7.0, and 8 to 10 units of galactose oxidase, which was purifiedby affinity chromatography (9), were added. The cells wereincubated for 5 min at 37° with occasional gentle mixing;
washed 3 times with PBS, pH 7.0; and treated with 500 /iCiNaB3H4 as described above. A number of controls were used
omitting neuraminidase, galactose oxidase, or both sialidaseand galactose oxidase.
Preparation of Surface Membranes
The labeling techniques used did not greatly reduce cellviability and had little detectable effect on the techniquesnecessary for the preparation and isolation of cell membranes.The zinc chloride method (39) was performed with a pretreatment with hypotonie ZnCI2 for 6 min at ambient temperaturefollowed by 6 min on ice. In the case of cells harvested with
disodium EDTA, a variation in the ZnCI2 treatment was foundto be necessary to give yields of whole membranes similar tothose obtained from trypsinized cells. To a suspension of 5 x107 cells in 1 ml of 0.16 M NaCI, 2.5 ml of 1 mw ZnCI2 were
added, and the cells were gently stirred and kept for 4 min atambient temperature. The cell suspension was then cooled inice for 6 min, with the addition of 2.5 ml of 10 mw ZnCI? after30 to 60 sec. In all cases of homogenization, it was foundpreferable to use a loosely fitting A pestle in the Douncehomogenizer for 50 to 100 strokes by hand, followed by anumber of strokes with a tighter fitting A pestle. The homogenization and purification procedures were followed by observation of the membranes in the phase-contrast microscope.
The resulting cell fractions, separated by sucrose density gradient centrifugation, were examined for radioactivity (35) andprotein content (28). A fraction of whole membranes, free ofnuclei and other particulate matter, was collected from the firstsucrose gradient (40). Another membrane fraction which wascontaminated with nuclei was purified by recentrifugation on agradient of 35 to 45% sucrose solutions.
Acid Hydrolysis
Tritiated oxidized sialic acid residues were detected andassayed by acid hydrolysis of labeled cells and membranesusing 0.1 N H2SO4 at 80°for 1 hr. Solutions were diluted with
water and neutralized with solid barium carbonate, and thesupernatant solutions, after clearing by centrifugation, werelyophilized and applied to Whatman No. 1 paper. After chro-matography overnight in n-butyl alcohol:acetic acid:water (5:2:2 or 3:2:1) or n-butyl alcohol-pyridine:1 N HCI (5:3:2), paperswere dried and 0.5-cm strips were cut for radioactive counting.
The silver nitrate dip reagent (36) was used to locate the sialicacid and other monosaccharide markers.
Radioactive galactose and galactosamine were characterized in a similar fashion using stronger acid conditions forhydrolysis (5 hr with 2 N HCI at 100°), and the acid was
removed by treatment with Dowex 1 (carbonate form). Chro-
matograms were developed in ethyl acetate:pyridine:water (12:5:4) and subsequently in n-butyl alcohohacetic acid:water (3:
2:1).
SDS:polyacrylamide Gel Electrophoresis
Electrophoresis on 7 to 14% gradient polyacrylamide slabgels in SDS was carried out essentially by the procedure ofLaemmli (26) in the presence of mercaptoethanol. Staining forproteins was by Coomassie blue. For ascertaining the distribution of radioactivity, the gels were dried and 1.5-mm strips
were cut, placed in scintillation vials, and prepared for countingradioactivity by the following procedure. Water (50 /il) wasadded to each vial and, after 10 min at ambient temperature,400 ¡i\of Protosol were added. After the blue stain eluted fromthe strips (30 to 60 min), 4 ml of PPO-POPOP solution intoluene were added, and the vials were incubated for 18 hr at37°before radioactive counting (25). The marker proteins and
ton Biochemical Corp., Freehold, N. J.); bovine serum albumin,M.W. 66,000 (Miles Labs, Inc., Elkhart, Ind.); ovalbumin, M.W.45,000 (ICN Pharmaceutical, Inc., Cleveland, Ohio); and chy-
motrypsin, M.W. 25,000 (Worthington Biochemical Corp. forPharmacia, Piscataway, N. J.).
Chemicals and Enzymes
NaB3H4 (6 Ci/mmol), L-[14C]fucose (50.8 mCi/mmol), and
Protosol were purchased from New England Nuclear, Boston,Mass. V. cholerae neuraminidase, galactose oxidase, and pro-nase were purchased from Calbiochem, La Jolla, Calif., andtrypsin (3 times crystallized) and soybean trypsin inhibitor werefrom Worthington. All chemicals were of reagent grade.
RESULTS
Cell Harvesting. Cells were removed from monolayers by avariety of methods. In all cases, it was found that the BHK2i/da cells dislodged more rapidly than their transformants. Inone case, BHK2i/d3 cells were removed rapidly with isotonicphosphate, pH 7.4, while the transformed cells were not removed by this procedure.
Periodate Oxidation. Maximum oxidation of 1 x 108 cells
was accomplished with 2 mwi sodium periodate in ¡sotonicphosphate, pH 7.4, within 10 min at room temperature withgentle mixing. Prolonged treatment with sodium periodate resulted in up to 20% loss of cell viability as measured by theuptake of trypan blue. At 5°,when maximum oxidation was
prolonged beyond 30 min, lower viability was observed again.For comparison, cells were treated with galactose oxidase withor without neuraminidase; again, any prolonged incubations,such as in the standard galactose oxidase procedures (10),resulted in up to 40% loss of cell viability. Particularly destructive to the cells was the prior incubation with neuraminidasenecessary to expose the galactose residues which are requiredfor the galactose oxidase to function. Therefore, mild conditions, 15 min with neuraminidase and 5 min with galactoseoxidase at 37°,were used to oxidize the cells.
Radioactive Labeling of Cells. The reaction of both celltypes (BHK21/C,3 or Ci3/B4) with sodium borotritide led toconsiderable uptake of nonspecific label (Table 1). The radioactivity persisted throughout the fractionation procedures, andthe purified surface membranes contained a reasonable proportion of the radioactivity which was directed to sialic acid.However, the fractions undergoing prior oxidation of carbohydrate groups, either by periodate or galactose oxidase, showed
Table 1
Distribution of radioactivity in cell fractions after borotritide labeling
higher radioactivity than did the controls which had not undergone the oxidation reaction. There was little enhancement ofradioactivity above the control when galactose oxidase treatment was given directly to the cells without previous desialy-
lation with V. cholerae neuraminidase.There was incorporation of 3H irrespective of the method of
harvesting the cells, whether through trypsinization, disodiumEDTA, or phosphate-containing buffers. The radioactivity in
corporated was always several times greater than the nonspecific labeling of control cells, even if the cells were labeled onmonolayers and then harvested (Table 2).
In all cases, cell viability was high (>90%), but it was foundthat a sharp fall in viability resulted if cells were incubated forlonger than 5 min with sodium borotritide, especially at roomtemperature. Excess sodium borohydride, added to ensurecompleteness of reductions, also led to a marked drop inviability of the cells. Therefore, incubation was only for 5 minat 5°,and no additional sodium borohydride was added afterthe reaction with NaB3H4.
Incorporation of Radioactivity into Membrane Fractions.All of the fractions which were separated on gradients ofsucrose solutions were analyzed for radioactivity and protein.All of the preparations of whole surface membranes appearedas shown in Fig. 1, A and B. Of special interest were thespecific activities of the purified membrane fractions expressedas percentages of whole cells before homogenization (Table3). With the exception of the BHK2i/Ci3 cells harvested withdisodium EDTA (Table 1), there was a concentration of radioactivity in the membrane fraction irrespective of the method ofharvesting the cells or oxidation prior to sodium borotritidereduction, so that the specific activity in this fraction exceededthat measured in the unbroken cells. The specific activity ofthe membrane fractions also exceeded that of the cytoplasmicfractions which were examined at the same time. On the otherhand, in the case of controls which had not been oxidized, thespecific activity in the membrane fractions was usually thelowest. This result emphasized the difference in uptake oftritium exhibited between oxidized and control cells. Therefore,the incorporation into the membranes was greater than into thecells in the former case, and the reverse was true in the controlcells.
Labeling of Isolated Membranes. The whole membraneswere labeled after removal of sucrose by several washings withthe requisite buffer and centrifugation at 8000 x g. Loss of themembranes occurred by this treatment and was due to vesi-cularization, which was observed in the phase-contrast micro
scope. Only the membranes which appeared whole, as shown
Table 2Ce//s labeled on the mono/ayer with NaB3H, after NalO, oxidation and then
harvested
Cell fraction (cpm/mgprotein)Cell
typeBHK?i
/Ci3BHK2,/C,3C,3/B40,3/6,Treated
cells191004Controlcells4
242Mem
branes35
3088
11Control
membranes2
1432Isolation
methoda
bt>
b
.
B '
.
Fig. 1. Surface membranes of Cis/B« cells prepared by the zinc ion technique. Phase-contrast microscope. A. x 125; 8. x 500.
Table 3
Specific activity of surface membranes harvested by different procedures
Sequence of treat-ment
Specific activity of membranes (% of whole cell)a-
BHK21/Ci3 C,3/B4 Comments
Trypsin-NalO. 162 316
NalCvtrypsin 440 164
NalCvno trypsin 184Galactose oxidase-no 158
trypsinEDTA-NalO. 111 197
EDTA-galactose 79 183oxidase
Trypsin-galactose 170 419oxidase
Exposed higher % for labelingin transformed cell.
More trypsin-sensitivematerial on the cell surfaceafter transformation
Prior treatment before labeluncovered higher % intransformed cell.
Consistent with above; trypsinexposed higher % forlabeling in transformed cell.
a After addition of N;ilO, and washing, the cells dislodged so that the reactionwith NaB3H«was with a cell suspension and not on a monolayer.
b Cells labeled as monolayers then removed by trypsin.
Specific activity was determined as cpm/mg protein of the cells or membranes.
in Fig. 1, were used. The specific activities (corrected for thecontrol membranes) were 989 and 1924 cpm/mg of proteinfor the membranes from BHK2i/Ci3 and C,3/B4 cells, respectively, and were higher than those obtained by labeling thewhole cell and isolating the membrane (Tables 1 and 2). This
result was to be expected when the membranes were isolatedfrom cells labeled prior to trypsinization since 20 to 30% of theradioactivity was removed by the trypsinization procedure. Itwas, however, unexpected when the cells were removed byother procedures such as those in Tables 2 and 3.
Characterization of Sugar Residues. The sugar residueslabeled in the membranes by borotritide were characterized bytheir rates of migration on paper chromatograms after acidhydrolysis. In the case of membranes from the periodate-oxi-dized cells, the only radioactive sugar observed was the 7-
carbon fragment of neuraminic acid, as shown by comparisonwith oxidized sialic acid standards. On the other hand, thegalactose oxidase-treated cells showed both [3H]galactose and[3H]galactosamine in a ratio of 4:1. No 3H-labeled migrating
sugar was found in nonspecifically labeled membranes, i.e.,those prepared from sodium borotritide treatment without previous oxidation. Less than 15% of the radioactivity was extracted into chloroform:methanol from the radioactive membranes, suggesting that the major portion of the radioactivecarbohydrate was not in lipid macromolecules.
Gel Electrophoresis. Electrophoresis of labeled membranepreparations in SDS on 7 to 14% gradients of polyacrylamidefollowed by staining with Coomassie blue did not reveal anydifferences due to the chemical reactions involved in oxidationwith either periodate or galactose oxidase. However, differences in the proteins of membrane preparations of the transformed cells and the normal counterpart which were reportedpreviously were observed (17). Small differences were alsoseen depending upon the harvest techniques, trypsinization, orother means of removal, although the majority of the proteinbands were similar.
On comparing 3H-labeled bands, a number of differences
were noted. The most apparent was that between the profilesof the membranes from the radioactive transformed cells andthose from the normal counterpart. Almost 40% of the radioactivity was found in glycoproteins from the transformed cellsin the area of the gel at M.W. 45,000 to 50,000, while thenormal counterpart contained similar material with a molecularweight of approximately 35,000 to 40,000 (Charts 1 and 2).This was reproducibly seen in all of the gels, regardless of theprior treatments. In addition, the radioactive glycoproteins fromthe transformed cells showed 2 prominent areas of radioactivity(Chart 2), while those of the normal counterpart had one areaof radioactivity at a molecular weight of approximately 40,000which was more predominant than the higher-molecular-weight
area (Chart 1).Some differences were also seen when the radioactive mem
branes which resulted from the different procedures werecompared. Trypsin not only shifted the profiles by reducingM.W. >90,000 material but also appeared to shift the proportions of the radioactive bands (Chart 1). That is, the pronouncedpeak of radioactivity seen in the M.W. 40,000 region was notas high in comparison with the rest of the radioactive areas(Chart 1, A and C). Proportionately more radioactivity wasobserved at M.W. >100,000 in the membranes which were notfrom the trypsinized cells (Chart 1, ßand D). A weakly radioactive peak at approximately M.W. 250,000 was revealed inmembranes from both cell types if the cells were not harvestedby trypsinization. In contrast, membranes from trypsinizedcells, either normal or transformed, failed to show this labeledglycoprotein to the same extent (Charts 1A and 2A).
300
Q_O
•¿�200
gg
T3¡Cce
100
93K 66K 45K 25K
lili
93K 66K45K 25K
lili
Nal04
45
Fraction Number90 45
Fraction Number90
100
CJ
g'-oIDce
50
10
93K 66K
I I
93K 66K 45K
45
Fraction Number90 45 90
Fraction Number
Chart 1 SDS:polyacrylamide gradient gel electrophoresis of radioactive surface membranes isolated from BHK21/Ci3 cells. Prior to membrane isolation, thecells were harvested by trypsinization (A and C) or with PBS, pH 7.0 (B and D),and labeled with sodium borotritide after oxidation with sodium periodate (A andB) or galactose (GaO oxidase (C and O). . nonspecific radioactivity of thecontrol membranes, which in all cases was the same. Details are described in"Materials and Methods." K numbers, molecular weight in thousands.
There were some differences between the profiles of theradioactive membranes from periodate-treated and galactoseoxidase-treated BHK2i/Ci3 cells (Chart 1). Additional 3H peaks
were present in the membranes from the galactose oxidase-
treated cells which were not as prominent in those treated withperiodate. The profiles of the membranes from Ci3/B4 cellslabeled after oxidation by either method showed no apparentdifferences (Chart 2, A and 8).
Isolated membranes, prepared from trypsinized Ci3/B4 cells,were also tritiated, and the SDSrgel scans were compared withthose labeled prior to membrane isolation. Again, a number ofdifferences were seen in the radioactive profiles (Chart 3). Theborotritide-treated surface membranes showed proportionately
more radioactivity above M.W. 150,000 (Chart 3) than did themembranes isolated from the borotritide-treated cells (Chart2). Other differences were seen in the proportions of the peaks
Chart 2. SDS:polyacrylamide gradient gel electrophoresis of radioactive surface membranes isolated from C,3/B* cells. The cells were harvested by trypsin-ization and radioactively labeled with sodium borotritide after oxidation withsodium periodate (A) or galactose (GaO oxidase (B), and surface membraneswere isolated from the radioactive cells. Details are described in "Materials andMethods." The control membranes always gave the pattern shown by the dashed
lines of Chart 1, C and O. K numbers, molecular weight in thousands.
O 50
10
A
NalO
93K 66K 45K 25K 93K 66K 45K 25K
45
Fraction Number90 45
Fraction Number90
Chart 3. SDS:polyacrylamide gradient gel electrophoresis of radioactive surface membranes from Cn/B« cells. Surface membranes were isolated fromtrypsinized ds/B« cells and subsequently were made radioactive by sodiumborotritide after oxidation with sodium periodate (A) or galactose (GaO oxidase(B). Details are described in "Materials and Methods." K numbers, molecular
weight in thousands.
between M.W. 70,000 and M.W. 110,000. In addition, theradioactivity between M.W. 40,000 and M.W. 50,000 wasalways seen as proportionately more in the M.W. 50,000 regionof the gel when the isolated membranes were labeled (Chart3).
SDS:gels of control preparations, i.e., membranes from cellswhich had been treated with NaB3H4 (a) without prior periodate
oxidation or (fa) without galactose oxidase treatment failed toshow the characteristic peaks present in the electrophoreticprofiles of oxidized cells. Instead, a single strong peak atapproximately M.W. 50,000 appeared and was presumablyindicative of the site of nonspecific 3H labeling of the surface
membranes. Similarly, membranes prepared from cells whichhad been incubated with galactose oxidase without previoussialidase treatment failed to incorporate tritium specifically, andthese gel scans were also limited to the M.W. 50,000 peak(Chart 1, C and D).
Glycopeptides from Labeled Cells. BHK..,/C,, cells whichhad been labeled on the monolayer by periodate-borotritide
were trypsinated, and the material removed by this procedure,the "trypsinates," contained 300 cpm//xg of membrane pro
tein. Since C,3/B4 cells could not be labeled directly on themonolayer, both cell types were removed with EDTA, labeledwith borotritide after periodate oxidation, and trypsinized. Ahigher percentage of radioactivity was incorporated into thetrypsinate of Ci3/B4 cells (25 cpm/jug of membrane protein)than into the BHK2i/Ci3 cells (11 cpm/ftg of membrane protein). However, the relative incorporation into the trypsinatescompared to the cells was similar (30 and 25%, respectively).Further digestion of the trypsinates with pronase and separation of the glycopeptides on Sephadex G-50 columns, after
EDTA treatment, showed profiles similar to those reportedpreviously (12, 16). However, both cell types contained significant radioactive material which was excluded from the gel incontrast to similar trypsinates metabolically labeled with radioactive fucose. Similar to previous reports, the trypsinates fromCi3/B4 cells had an abundance of material which eluted morerapidly from the Sephadex column when compared to that ofthe nontransformed cells.
DISCUSSION
A method has been devised to specifically label sialic acid-
containing glycoproteins on the surface of cells in culture. Themethod is rapid and involves very little perturbation of the cell;2 treatments are required, one with sodium periodate for 10min at ambient temperature and another with sodium borotritidefor 5 min at 5°.The fact that the label was enriched in the
surface membrane was shown by isolation of purified preparations of whole surface membranes (Fig. 1). No alteration ofthe surface membrane was seen in terms of isolability orproteins, as shown by Coomassie blue staining after electrophoresis on polyacrylamide gels. Moreover, the method wasapplied successfully to 2 cell types, virus-transformed baby
hamster kidney fibroblasts and the normal counterpart.A number of differences were noted when the sialic acid-
containing glycoproteins of the hamster kidney fibroblast membranes were compared with those of their transformants. Thepresence of glycoproteins of an apparent higher molecularweight in the transformed cells was particularly striking, aswere the number of different species (Charts 1 and 2). Inaddition, other higher-molecular-weight species were revealed
when the isolated membranes were labeled and compared tothose labeled on the whole cell (Chart 3). These glycoproteinsmay be those which span the membrane (34) or cytoskeletalelements which appear as fuzz on the cytoplasmic side ofisolated zinc ion-treated membranes (14). This material has
been shown to contain several major proteins (29).The specific activity of the reduced sialic acid was similar in
the surface membranes from both periodate-treated nontransformed and transformed cells, although the BHK2i/Ci3 cellshad high specific activities in the whole cells and cytoplasm(Table 1). This cautions against the use of whole cells andunpurified membranes to draw conclusions about the membrane. In accord with the results of Juliano and Behar-Bannelier(23) using other surface labeling procedures, cell fractionationis necessary in order to insure detection of membrane proteins.A number of nonviable cells take up the reagents or alternately
are lysed and their contents take up the reagents. Monitoringby electron microscopy cannot be used to eliminate this artifact(1), inasmuch as the atypical cells which are internally labeledand contribute to the radiochemical analyses cannot be detected (24).
The periodate-borotritide method marked some different gly-coproteins in BHK2i/Ci3 fibroblasta than the well-known pro
cedure utilizing galactose oxidase after neuraminidase treatment (11). The additional incubation periods (20 min at 37°)
required of the latter procedure could allow the exposure ofdifferent glycoproteins on the surface during this time. It shouldalso be pointed out that longer incubations reduced the viabilityof the hamster cells as much as 40%.
Previous studies have used periodate oxidation coupled withborotritide reduction to label erythrocytes (27, 30), and in arecent report it was used to label lymphoid cells (8). In thelatter case, Gahmberg and Andersson (8) treated the cells withNaB3H4 for 30 min at room temperature, whereas milder conditions are reported here (5 min at 5°). Such a prolongedNaB3H4 treatment led to a high background of nonspecific label
and loss of viability for the hamster cells. Moreover, the isolatedsurface membranes of the hamster fibroblasts had increasedspecific labeling many times over that of the oxidized cells. Itwould be interesting to know the percentage of incorporatedradioactivity which was specific to the surface membranes oflymphoid cells (9) rather than the internal portion.
In the experiments reported here, BHK2i/Ci3 cells had theexpected morphology of a normal cell, and the transformantshad a rounded appearance. In all the methods used for dislodging the cells from the plastic surface, which includedtrypsin, EDTA, PBS, and isotonic phosphate, the normal hamster cells were dislodged more readily than were the transformed cells. Indeed, PBS alone was insufficient to removetransformed cells to any extent, while complete removal ofBHK2i/C,3 cells was attained after incubation for only 15 to 20min. The differential extraction of the cytoskeletal proteins (29)may account partially for these variations in dislodgement. Theresistance to detachment by all of these methods supports theobservations of Juliano (22), which suggested that adhesiveness and cell shape are not necessarily related. In addition,since there is a relatively small amount of fibronectin (37)present in the BHK2i/Ci3 cells which are transformed, it canbe deduced that the differential attachment of these cells tothe plastic is not widely controlled by the fibronectin present(20), but by other factors (31). For example, it has beenreported that cell lines which were deficient in terminal saccha-rides adhered less well than normally (32). Conversely, theantennary structure of the branching terminal oligosaccharidesmay not be related to cell adhesion as conflicting results wouldsuggest (22). However, our findings that the transformed cellswith highly branched surface glycopeptides (16, 35) adheredmore strongly than did the normal counterpart support theconcept (32) that the more glycosylated the cell surface glycoproteins, the stronger the adhesive forces (41).
Prior treatment to detach the cells with EDTA or trypsinincreased the specific activity of sialic acid in the transformedcell membranes over that of the whole cell, whereas the specificactivity of BHK21/Ci3 cell membanes was not increased to thesame extent. It is possible that these treatments to detach thecells uncover additional sialic acid residues in the transformedcells or, conversely, remove more nonlabeled protein which
would give the increased specific activity. In contrast, labelingof the cells prior to trypsinization increased the specific activityof sialic acid in the membranes from BHK2,/d3 cells while thetotal radioactivity per mg protein, as well as the specific activity,was dramatically reduced in the transformed cells. When surface membranes were prepared from the trypsinized cells andsubsequently labeled, the amount of radioactivity incorporatedper mg protein of d3/B4 membranes was twice that of theBHK21/Ci3 membranes. Again this suggested that trypsin exposed more sialic acid residues in the transformed cell.
Most of the external membrane glycoproteins in the hamstercells contained sialic acid and fucose in that similar polyacryl-
amide gel patterns were seen when the cells were metabolicallylabeled with fucose and externally labeled with borotritide afterperiodate oxidation. By using this reasonably gentle method,the artifacts of harsh treatments are avoided, and it is nowpossible to doubly label membrane glycoproteins and to examine further the relationship of the reported differences totransformation.
ACKNOWLEDGMENTS
The excellent technical assistance of Ray-Jen Chang, Jean Kershaw, andFlorence Massey is acknowledged.
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