THE JOCRXlL OF I~IoLoCICAL CHEMISTRY

Vol. 246, No. 20, Insue of October 25, pp. 6339-6346, 19il
hinted in U.S.A.

Glycoproteins of Cell Surf aces

A COi\\Il’ARATIVE STI’DY OF THREE DIFFERENT CELL SURFACES OF THE RAT

(Received for publication, May 24, 1971)

HARTMUT GLOSSMANN” AND DAVID X1. NEVILLE, JR.

From the Section on Physical Chemistry, Laboratory of Neurochemistry, National Institute of Mental Health,
Bethesda, Maryland 20014

SUMMARY recent finding of concanavaliu A sites in embryonic tissue (8)

suggests that cell transformation involves in part a dedifferen-
The glycoproteins of three different cell surface membranes
tiatiou of the cell surface. Since embryonal antigens are preseilt
of the rat have been investigated by acrylamide gel electro- in tumor tissue (9), one may ask whether or not retrogenetic

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phoresis with the use of discontinuous buffers in the presence
expression is a cause or an effect of carcinogenesis.
of sodium dodecyl sulfate (SDS). Liver, kidney brush bor-
Further support for a role of glycoproteins in growth regulat,ion
der, and erythrocyte ghost membranes are solubilized in SDS
comes from observed differeilces in glycopept’ides derived from
and after brief exposure to dilute base are reduced with /3-
cell surfaces of virus transformed and noutransformed cells
mercaptoethanol. The gels, after washing free of SDS, are
(10). Since similar results were obtained with cells trailsformed
Schiff-stained, and it is demonstrated, by the use of glyco-
with polyoma virus and Rous sarcoma virus, the additioilal or
proteins of known molecular weight and subunit composition,
altered glycoproteins in the surface membrane are probably not
that the stained patterns represent size separations of glyco-
coded by the virus genome (10).
protein subunits. Each membrane is found to have an iden-
Histocompatibility seems to be determined by glycoproteins
tifiable glycoprotein subunit pattern composed of at least 6 to
on the cell surface. For the mouse II-2 alloautigens, it was
11 different sized subunits. Most of the staining intensity is shown that the activity is located on glycopeptides derived
present in one to three subunits. Although each membrane
from a papain-digested crude membrane fraction (11). The
has its own unique pattern, liver and kidney brush border
HL-A-alloantigens derived from human lymphocyt8es by papain
have four identically sized subunits. These subunits have
digestion are also glycopeptides and contain 7 to 8% carbohy-
molecular weights of 250,000, 195,000, 130,000, and 96,000
drate (12).
when estimated by interpolations of relative mobilities be-
A role of glycoproteins in specific cell adhesion is also suggested
tween protein markers. The erythrocyte ghost also contains
by findings by Moscona (13).
a 96,000 molecular weight glycoprotein subunit. Sialic acid
Oppenheimer and coworkers (14) have presemetl evidence
determinations made on cut gel slices show that all promi-
t’hat intercellular adhesion seems to depcud on an incorl)oration
nent Schiff -positive glycoproteins contain sialic acid. of sugar in macromolecules in t.he cell surface. One can ad-
sume that at least part of the normal cell recognition and cell
adhesion would (a) be an active metabolic process and (b) re-
quire glycoproteins. The present investigation was designed
to acquire information about the glycoproteins iu purified cell
membranes without resorting to prior treatment with proteases.
Carbohydrates in cell surfaces represent only a minor fraction
but play an important role in regulation of cell growth, anti- MS TERIA LS
genicity, and cellular recognition.
N-Acetyhleuraminic acid, glucosamine, galactosamine, trails-
Since most of the carbohydrate in cell surfaces is bound to
ferrin (human, ion-free), and avidin (egg white) n-ere obt,ained
protein, interest has focused on membrane glycoproteins. How-
from Sigma. Fetuin (Spiro method) was obtained from Grand
ever, only the membrane glycoproteins of the human red blood
Island Biological Company (Grand Island, Sew York). 01,.
cell ghost carrying M and N blood group specificit,y are well
Glycoprotein (human) and thyroglobulin (pig) were obtained
characterized (l-3).
from Schwarz BioResearch. Chorionic gouadotropin (human
During mitosis (4) and after cell transformation (5, 6), carbo-
pregnancy urine), about 7500 to 9000 i.u. per mg, aud cerulo-
hydrate side chains of the cell surface can react with plant ag-
plasmin were a gift from Dr. Hickman (Xational Institutes of
glutinins (concanavalin A, wheat germ agglutinin). Uurger
Health). Glucose oxidase (Aspergillus niger) was obtained
and Noonan (7) could restore contact inhibition to transformed
from Worthington. 8-Anilinonaphthalenesulfonic acid (mag-
cells by treatment with a monovalent concanavalin A. The
nesium salt) was obtained from Serva (Heidelberg, Germany).
* Visiting Associat,e sponsored by Delitsche Forschungsgemein- Experimental Animals-Male rat,s of t’he Sprague-Darvley
schaftj. strain were used for the three different membrane preparations.

6339
 6340 Glycoproteins of Cell &r.fuces Vol. 246, No. 20

METHODS The gels were prepared and stained with Coomassie blue as
IlfeuLbrane Preparatiorb-Plasma membranes from rat liver described in the preceding paper (27).
were prepared as described elsewhere (15). Kidney brush Schi$ Staining-None of the published procedures, especially
border was obtained by the method of Wilfong and Xeville the commonly used method of Zacharius et al. (28), worked
(16). Red blood cell ghosts were prepared according to the satisfactorily in the SDS-containing gels. The following method
method of Dodge, Rlitchell, and IIanahan (17) with 1000 i.u. was worked out to elute interfering SDS from the gel, including
of heparin per 100 ml of blood as an anticoagulant. Freshly SDS bound to protein, and to keep the gel at acid pH during
drawn blood was cooled down immediately to 2”, and the every step of the staining reaction in order to avoid simple acid-
erythrocyt,es were washed five times with isotonic phosphate base staining. Controls of gels without periodic oxidation
buffer, pH 7.2. The Huffy coat was carefully removed by and gels containing well characterized glgcoproteins (ovalbumin,
aspiration. After lysis, the ghosts were washed only two times. fetuin, ovomucoid, human a,-glycoprotein, and tjhgroglobulin)
All membrane preparations were done between 2-4”, and mem- and proteins (albumin, hemoglobin, chymotrypsinogen Aj and
branes \vere eit’her used immediately after preparation or quick myosin) were always performed. As outlined later, only with
frozen and stored at -70” until used. the procedure described below could artifactual staining of some
Chemical Determinations-After precipitation with 1 y0 phos- noncarbohydrate-containing proteins be avoided.
photungatic acid in 0.5 x HCl and washing two times with 5% The following procedure is a combination of a m-ashing step
trichloroacetic acid, the membrane pellet was dried overnight and a staining procedure for carbohydrates as described bs
at room temperature in a desiccator containing KOH and Maurer (29).
anhydrous CaS04. For protein determination, the pellet was 1. Twelve gels were washed continuously with 2.6 liters of
40% methanol and 7% acetic acid overnight (destainer from

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dissolved in 0.1 N NaOH, and analyses were performed with
aliquots according to the method of Lowry et al. (18). Crystal- Hoefer Scientific Instruments, San Francisco, California). The
line serum albumin served as a standard. Sialic acid was de- solution was changed and clution continued for 8 hours.
termined after 1 hour of hydrolysis in 0.1 N HzS04 at 85O wit.h 2. Gels were oxidized for 1 hour at 4” with 1 y0 periodic acid in
thiobarbit,uric acid according to the method of Aminoff (19). 7% acetic acid in the dark.
X-Acetylneuraminic acid was the standard. For delipidntion 3. Gels were washed in a destainer containing 7% acetic acid
the pellet was suspended in 0.5 ml of methanol. after adding 1 over a period of 24 hours; the solution was changed two times.
ml of chloroform, the suspension was incubated 2 hours at 45” 4. Gels were incubated for 1 hour in Schiff’s reagent at 4” in
and centrifuged 4 min at 12,000 x g. This step was repeated the dark.
twice, and the dried collected supernatants were analyzed for 5. The gels were washed three to four times with 1 y0 Na&06
cholesterol (20) and inorganic phosphorus after digestion with in 0.1 N HCl and stored in this solution or in Schiff’s reagent.
10% l\IgXOi in absolute et,hanol for 1 hour at 200”. Inorganic Since it was not possible to determine which band in the
phosphorus was determined with the method of Ames and Schiff-stained gels corresponds to a band in the Coomassie-
Dubin (21). KzHI’O.1, dissolved in 0.1 N sulfuric acid, served stained gels because of the complicaated pattern and the slightly
as a standard. Neutral sugars in delipidated membranes were different swelling of the gels, the ability of 8-anilinonaphthalene-
determined with the anthrone method (22) with D-glucose as a sulfonic acid (A%) was used to visualize the protein bands after
standard. Amino sugars were det#ermined after hydrolysis with ANS Schiff stain (30).
2 N HC’l at, 100” for 16 hours, according to the met’hod of Gatt ANS (magnesium salt) (10 mg) was dissolved in 100 ml of 1 y0
and Berman (23). Galactosamine hydrochloride served as a KazSa05-0.1 N IICl. Gels were incubat)ed in this solution 1 to 2
standard. hours. After washing with lyO Na&05-0.1 N IICl, the gels
Xolubiiization of Xembranes-To 1 ml of membrane suspen- showed a brilliant fluorescence of protein bands Imder ultra-
sion containing about 5 mg of protein, solid Ka,CO, was added violet light’, and an exact localization of the Schiff-staining bands
(2 mg of N&&O3 per mg of protein) to bring the pH to 11. was possible. Determinat,ions of apparent molecular weights
Solid SDS1 was added (8 mg per mg of protein). After 1 to 2 were done as described (25) by comparing relative mobilities
min, 2-mercaptoethanol was added dropwise with gentle shaking with marker proteins and calculating apparent molecular
to bring the total concentration to 10% and drop the pH to 8.5. weights by interpolat’ion between two marker proteins on a
‘l’hc dissolved membranes were stored anaerobically in Thun- standard calibration curve.
berg tubes at -20” or used immediately. IZESULTS
Before electrophoresis, solubilized membranes were dialyzed
4 to 15 hours at 4” against a solution of 2 III deionized urea, 0.05% Chemical Corrzposition of Membranes- -The chemical composi-
dithiothreitol, and O.lyO SDS in upper gel buffer in order to tion of t’he different membranes studied is given in Table I.
equilibrate t.he ionic composition of t.he sample to the upper gel Washing the membranes several tirnes with distilled water in-
and to dimiliish the amount of sucrose in liver membrane and stead of with phospklotungstic acid and t’richloroacetic acid failed
kidllc;v bruskl border present after gradient centrifugation. to remove traces of sucrose which were present in membrane
Discontirvmolrs Electrophoresis--Two discontinuous SDS elec- fractions after gradient centrifugation and which interfered with
trophoresis systems were used: a sulfate-borate system calcu- the assays. @albumin was used as a control in the determina-
lated by Jovin, Dante, and Chrambach (24) and modified by tion of tobal sugars and hexosamines. The result,s are in good
Xcvillr (25) for SDS (running pH 9.5) and a modification of the agreement with the published values (1.2 y0 glucosamine and 2 y0
Ornstriri-Davis system as described (26). mannose (31)). It’ should be noted that the red blood cell
ghost preparations still contained hemoglobin. Since repeated
1 The abbreviations used are: SDS, dodecyl sulfate sodium washing of ghosts removes loosely bound membrane proteins
salt; ASS, 8.,znilinonaphthalenesulfolzic acid (magnesium salt). (321, a 10% contamination with hemoglobin was tolerat~ed.
Issue of October 25, 1971 H. Glossmann and D. M. Neville, Jr. 6341

TABLE I
Chemical composition of membranesa

Total hexoses* HWXtllheSC Sialic acidd Cholesterol Phospholipidd

pg,mg protein ?imoles/mg proteia pgg/mg ~roteilz

Red blood cell ghost. ............. 64 f 8 27 f 3 61 rt 10 177 f 10 483 f 41
Liver cell membrane. ............. 45 f 3 8&l 27 f 7 143 f 20 447 f 60
Kidney brush border. ............. 37 f 4 27 f 2 34 f 5 155 f 10 678 f 75
Ovalbumin ....................... 21 & 2 12 f 0.2 0 0 0

0 Each value is a mean value from four determinations.

* n-Glucose as standard; ovalbumin with n-mannose as standard.
c Galactosamine as standard.
d N-Acetylneuraminic acid as standard.
6 Calculated by multiplying lipid phosphorus (pg) by 25.

FIG. 1 (left). SDS electrophoresis of cell surface proteins. The
numbers indicate glycoproteins which are listed in Table II. A,
red blood cell (rat) (35 pg of protein); B, liver cell membrane
(rat) (55 pg of protein); C, kidney brush border (rat) (55 Mg of
protein); D, liver cell membrane (rat) incubated for 2 hours at
37” at pH 5.2 (0.2 m of sodium acetate buffer containing 100 I.E.
of penicillin and 50 pg of streptomycin per ml); E, kidney brush
border (rat) incubated as D. Arrows indicate position of glyco-
proteins.
FIG. 2 (right). Red blood cell ghost (human), 150 pg of protein,
stained for carbohydrate according to Zacharius (28). Arrows
indicate artifactual stained bands. Gel has different swelling
than B. B, red blood cell ghost (human), 150 pg of protein stained
for carbohydrate as indicated under “Methods.” The major Downloaded from http://www.jbc.org/ by guest on November 8, 2017
glycoprotein is a closely spaced doublet just below a very intense
Coomassie staining band which corresponds to a similar band in
the rat red blood cell ghost (Fig. 14, numbered 5). The sharp
dark line near the bottom of the gel is not Schiff positive, but a
whitish precipitate appearing dark in this reproduction. C,
human transferrin (20 pg) stained with Schiff’s reagent; D, fetuin
(calf) (35pg) stained with Schiff’s reagent; E, kidney brush border
(rat) (2OOpg of protein) stained with Schiff’s reagent; F, red blood
cell ghost (rat) stained with Schiff’s reagent (150 pg of protein);
G, liver cell membrane (rat) (200 pg of protein) stained with
Schiff’s reagent; H, same gel as G counter stained with ANS under
ultraviolet light.

Nevertheless, ghosts contained more sialic acid and total hexoses enough to detect about 0.5 /.~g of carbohydrate (measured with
than liver cell membrane and kidney brush border. ovalbumin) in a band. Since loads from 100 to 1000 +g of
Schiff Staining of Glycoproteins in Sodium Dodecyl Sulfate Gels membrane protein were used per gel, one should be able to detect
-The sensitivity of the Schiff staining procedure was high glycoproteins which represent about 1% of the total membrane
protein and contain 576 carbohydrat’c. Unless most of the three glycoproteins stairred hravily. One of these, with an ap-
SDS was i emoved before Scliiff smining, no reproducible results parent molecular weight, higher than 300,000, was found neither
KWC obtained. Depending ou the time a gel was incubated in in liver cell membrane nor in red blood cell ghost. Two rather
Schiff’s reagent and to what extent the washing after the periodic broad bands with apparent molecular weights of 130,000 and
acid oxidation was made, a deep red-colored background was 96,000 are prominent bands too. Two bands, charactcrist,ic for
obtainrd. After destaining for several days in 5% acetic acid, the SDS electrophoresis pattern of the cell surface proteins
marker proteins which contained no carbohydrate (hemoglobin, (apparent molecular weights of 250,000 and 195,000), were also
myosin, et,c.) were fouud to be stained. Since staining of pro- stained with Schiff stain.
teins occurred even ~vhcrr the periodic acid oxidation step was Liver Cell Men&une-The liver cell rncmbrane has fewer sialic
omitt’ed, the method of Zacharius et al. (28) and rclatcd methods acid and protein-bound hexosamincs and only slightly more pro-
which do not contain a washing step designed to eliminate tein-bound hexoses t,han the kidney brush border. This is also
protein-bound SDS can lead to artifactual smining in SDS- reflected by a more simple glycoprotein pattern of the liver cell
containing gels. membrane (Figs. 1 and 2 and Table II). The two prominent
The most likely explanation for this is a simple acid-base glycoproteins have apparent molecular weights of approximately
st,aining mechanism (33) of the Schiff leucobase reacting with 130,000 and 96,000. In mixing expcrirncnts, these bands co-
SDS still bound to prot,cin in the gels. An example of an electrophorese together with the corresponding bands in kidney
artificially stained gel ia given in Fig. 26. brush border. However, the resulting bands u-hen examined by
Uernbmne Glycoproteins-The pattern of Schiff-positive Schiff stain are broader, and it cannot be concluded that they arc
bands of the three different membranes was typical and repro- identical in size. As in the kidney brush border, the 250,000 and

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ducible. No difference in the pat,tcrn was noted under the fol- 195,000 bands were both stained with Schiff’s reagent. These
lowirrg conditions: (a) membranes solubilized immediately after bands coelectrophorcse wit,11 the corresponding bands in kidney
isolation in 5y0 SDS-lo% 2-mercaptoethanol (v/v), pH 11; (b) brush border. No splitting or broadening was observed in mix-
membranes stored at -70” up to 3 months and solubilized after ing experiments, and one can assume that they are identical in
thawing in 5y0 SDS-lo% 2-mercnptoethanol (v/v), pH 11; (c) size in both membranes.
membranes solubilized by heating 3 min at 90” in 3y0 SDS, 0.01 Red Blood Cell Ghost-The red blood cell ghost of the rat shows
M sodium phosphate buffer, pH 7.0, and 1O70 2-mcrcaptoethanol a completely different glycoprotein pattern. In contrast to
(v/v); (d) membranes solubilized and dialyzed as described un- kidney brush border and liver cell membrane, where all promi-
der %Iethods.” nent glycoproteins have apparent, molecular weights higher than
nIateria1 running with the buffer front’, which reacted with about 90,000, the prominent glycoproteins of ghosts have appar-
Schiff stain, was observed for each membrane. This material is ent molecular weights of 78,000, 70,000, 41,500, and 39,000.
probably identical with a rapidly moving material iu neutral SDS Another interesting feature of these glycoproteins, in contrast
elcctrophoresis, which was shown to be glycolipid and lipid to liver cell membranes and kidney brush border, was their al-
(34, 35). The glycoprotcin pattern was identical for both SDS most total lack of staining with Coomassie blue (gel loads, 25 to
gel systems, eliminating the possibility of artifact bands by 50 ,LL~of total protein). This property indicates that the glyco-
forming a complex wihh carbohydrate side chains and the borate proteins have a high carbohydrate content, as will bc outlined
ion (36) in the sulfate-borate system. later.
Kidney Bruslz Border-The kidney brush border has the most The human red blood cell ghost, which was examined for
complicated pattern of glyeoyroteins (Figs. 1 and 2 and Table comparison since the major glycoprotein is isolat,cd and charac-
II). About 11 bands stained with Schiff’s reagent, but only terized (l-3), had only two major glycoprotcins of nearly identi-

TARLE II
Appurent molecular weights of ylycoprofeins in membranes obtained by sodium clodecyl sulfate elecfrophoresis

Red blood cell membrane Liver cell membrane Kidney brush border

T
Band
number”

1
MOleCUlaI
weight

>250,000
RemarksC Band
number”
Molecular
weight

> 2.50) 000

Remarks
_- Band
numbe#

1
Molecular
weight

>300,000
Remarks

2 >230,000 >250,000 2 > 230,000

3 250,000 2;iO, 000 3 230,000
4 170,000 193,000 I lS.i,OOO
.J 96,000 130,000 P 5 130,000
6 78,000 96,000 P 6 96,000
7 70,000 7 76,000
8 68,000 8 70,000
0 41,500 9 65,000
10 39,000 10 60,000

a See Table TTI and Fig. 5 for validity of these values.

- I 11 .58,000

* The numbers refer to the numbering of glycoproteins in Fig. 1.

c P, prominent band; S, band with similar mobility present in serum; n, broad band, average molecular weight of 96,000, with a
leading edge of 92,000 molecular weight and a trailing edge of 98,000.
Issue of October 25, 1971 H. Glossmannand D. M. Neville, Jr. 6343

cal molecular weight and Schiff-staining intensity (apparent membrane has largely disappeared. The glycoproteins appear
molecular weight 82,000 and 78,000). In addition, two faint more resistant to destruction than the noncarbohydrate-contain-
bands with apparent molecular weights of 41,000 and 39,500 and ing proteins. This is in contrast to the red blood cell ghost
a faint band with an apparent molecular weight of 20,000 were (human and rat) in which no change in t.he protein or glycopro-
observed. Again, only very faint st,aining with Coomassie blue tein pattern after incubation up t.o 20 hours at 37” at pH 7.2 or
was observed, and exact localization of these glycoproteins was 5.2 occurred.
only possible with the ANS technique. Behavior of Various Glycoprofeins in Xodium Dodecyl Sulfafe
Chemical Determination in Gel Slices-When gels containing Electrophoresis-Since the behavior of glycoproteins with more
separated membrane proteins were sliced and examined for than 5 to 8% carbohydrate in SDS electrophoresis has not been
sialic acid, a typical pattern for each membrane was obtained
(Fig. 4). As can be seen from comparing Figs. 2 (E to G) and 4,
sialic acid was detected in all of the prominent Schiff-positive
bands. Again, the predominance of higher molecular weight
glycoproteins in liver cell membrane and kidney brush border
compared to red blood cell ghost is obvious. Although the
major glycoprotein in the kidney brush border (aljparent molecu-
lar weight >300,000) stained more intensely with Schiff stain
than the 130,000 and 96,000 bands (Fig. 2E), it contained less
sialic acid than the 96,000 band (Fig. 4). The spectrum of the

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chromophore was identical with the spectrum described by
Warren (37) (Fig. 3), and no interference of acrylamide gel or of
ANS was observed.
Mixing Experiments with Serum-The possibility that some of
the prominent glycoproteins in the membranes are contaminants
from serum was excluded by mixing experiments. Only in the
red blood cell ghost (rat) did a faint band with an apparent
molecular weight of 170,000 co-electrophorese with a major
glycoprotein in rat serum.
Peptidase Activity in Membranes-Both liver cell membrane
and kidney brush border contain peptidase activity, and great
care was taken during isolation, solubilization, and storage of
these fractions to avoid possible cleavage or degradation of
membrane proteins and glycoproteins. The protein pattern of
incubated membranes is given as an example in Fig. 1. After 2
hours of incubation at 37” at pH 5.2 under sterile conditions, the
normal pattern of proteins of kidney brush border and liver cell

I 3 1 I I ’
0 100 ”

WAVELENGTh (mpl
FIG. 4. Sialic acid det,ermination in gel slices after separation
of membrane proteins with discont’inuous SDS electrophoresis.
FIG. 3. Sialic acid determination in gel slices. Spectra of (Bottom of the gel is on the right.) Levels below 0.1 nmole per
chromophores extracted with acid butanol after thiobarbituric slice represent background color (Fig. 3~). The width of each
assay (Cary 15, l-cm cuvette, 0.1 O.D. range). (a) N-acetyl- column represents the width of the slice which was cut out. A,
neuraminic arid (2.5 nmoles); (b) Gel slice containing 96,000 kidney brush border (150 pg of protein); B, liver cell membrane
band of kidney brush border (Fig. U); (c) acrylamide gel slice (100 sg of protein); C, rat red blood cell ghost (100 fig of protein).
(1.5 mm) containing protein bound ANS; (d) diluted sample of The amount of sialic acid present in the buffer front was det’er-
ovomucoid; (e) acrylamide gel slice (1.5 mm) containing protein mined for red blood cell ghost (load 350 pg of protein) and is indi-
but no ANS; (j) acrylamide gel slice (1.5 mm). cated by the column on the fur right in C.
6344 Glycopyoteins of Cell Surfaces Vol. 246, h-0. 20

TABLE 111

Apparent molecular weights of glycoproteins obtained by sodium dodecyl sulfate electrophoresis

- I
Carbohydrate Mol wt by SDS
Glycoprotein Groupa Mel wt content electrophoresis
Deviation
-

Thyroglobulin (pig) I 669,000 (39) 335,000 (39) 8% (40)

Avidin 68,300 (39) 15,600 (41) 0% (41)

Geruloplasmin (human) II 60,000

16,000 (42) 8% (39) 135 ) 000 See text
95,000
55 ) 000
b

olr-Glycoprotein (human) II I 44,100 (39) -1lT, (39) 45,000 SF%

Chorionic gonadotrophin (hu- 30,000 (39) 30% (39) 34,000 +13s
man)
Ovomucoid 29,000 (38) 257" (39) %, 000 +20%
Fetuin (calf) 50,500 (39) 23~~ (39) 67,000 +330/,
Glucose oxidase (A, niger) 153,000 (43) 16% (44) 80,000
78,000 See text

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Transferrin 82,000 (45) 6% (39) 72,000 -12%
- -I -
Q Group I, glycoproteins with subunits identical in size; Group II, glycoproteins with subunits of different size Group III, glyco-
proteins existing as monomers.
* Additional band with molecular weight of 17,000 not staining with Schiff’s stain.

100 ovomucoid, lysoayme (38), and in the case of crl-glycoprotein,

r
probably albumin), and the glycoproteins could only be detected
with the Schiff stain.
Nobility of Clycoproteins in Sodium Dodecyl Sulfate Gel Elec-
trophoresis-The relative mobility in a gel for most of the tested
glycoproteins decreased with increa,sing molecular weight. After
plotting -100 log RF against molecular weight, a similar relation-
ship as described for proteins was obtained (Fig. 5). The rela-
tion between molecular weight (range 15,000 to 100,000) and
0 I I I / I I - 100 log relative mobility could be described by a least squares
20 40 60 80 100
MW ~10.~
line. The correlation coefficient for a set of seven marker pro-
teins was 0.9966 and for six glycoproteins 0.9284. The standard
FIG. 5. Relation between - 100 log RM (relative mobility) and
error of estimate of -100 log RF on molecular weight was 11.85
molecular weight for proteins and glycoproteins in SDS gels.
Proteins (0) : Hemoglobin, trypsin,
for proteins and 15.94 for glycoproteins, reflecting greater scat-
carbonic anhydrase, oval-
bumin, catalase, albumin, phosphorylase a. Glgcoproteins (0) : tering. When the relative mobilities of the glycoproteins in
(1) avidin, (2) ovomucoid, (3) human chorionic gonadotrophin, SDS gels were used to calculate apparent molecular weights from
(4) olr-glycoprotein (human), (5) fetuin (calf), (6) transferrin a calibration curve obtained with marker proteins (as was done
(human).
with membrane glycoproteins), deviations are observed (Table
III). The apparent molecular weights of fetuin, chorionic
previously reported, experimelnts were performed with purified gonadotrophin (human), ovomucoid, and avidin were found to
glycoproteins. Glycoprot’eins were dissolved in the same way as be higher than expected. However, ol,-glycoprotein (human),
membranes, run together with marker proteins, stained with thyroglobulin (pig), and transferrin (human) gave apparent
Schiff’s reagent, and the molecular weights estimated by compar- molecular weights which are in good agreement with the pub-
ing the mobility of the Schiff-positive bands with marker pro- lished results. According to the literature (listed in Table III),
teins after visualization with ANS. Electrophoretic mobilities the glycoproteins tested can be divided into three groups: (a)
of glycoproteins in gels must be assayed by Schiff staining rather glycoproteins existing as oligomers in solution with subunits
than protein staining if errors are to be avoided. It was not identical in size (avidin, thyroglobulin); (b) glycoproteins exist-
possible to stain most of the glycoproteins listed in Table III ing as oligomers in solution with subunits of different size
with Coomassie blue when applied in amounts between 1 and 15 (ceruloplasmin) ; (c) glycoproteins existing as monomers in solu-
~g per gel, and the possibility of error is high when one relies tion (al-glycoprotein, ovomucoid, transferrin, chorionic gonado-
only on a Coomassie-stained gel. Two examples may illustrate trophin, fetuin, and glucose oxidase). Each of the glycoproteins,
this. oclmGlycoprotein (human) separated on SDS gels, demon- with the exception of ceruloplasmin, moved as a single major
st,rated only one band with a molecular weight of 68,000, and band (or in the case of glucose oxidase as a closely spaced dou-
oromucoid (egg hen), only one band with a molecular weight of blet), and the apparent molecular weights found correspond to
14,500, when stained with Coomassie blue. The Cooma,ssie- the molecular weights for the monomers or subunits with the
stained bands represent minor contaminants (in the case of exception of glucose oxidase (A. n.iger). The results obtained
Issue of October 25, 1971 H. Glossmannand il. M. Neville. JY. 6345

with glucose oxidase and ceruloplasmin are reported in more relative mobility of glycoproteius and the molecular weight in a
detail. gel is high, and although the scatter is greater than that for pro-
Cerzcloplasmin-Ceruloplasmin had a complicated pattern (see t’eins the method seems, in general, valuable for estimating
Table III), and the question arises whether the bands with ap- molecular weights if errors as high as about 307, can be accepted.2
parent molecular weights of 95,000 and 135,000 are aggregates of For example, we fiud a molecular weight of about 80,000 for
subunits or contaminants. When ceruloplasmin is reduced and glucose oxidase (A. niger), although the literature reports 153,000
aminoet,hylated and fractionated on Sephadex G-200 in 5.5 M for the native enzyme (43). Since the enzyme binds 2 moles of
guanidine hydrochloride, three peaks with molecular weight’s F,4D (46), we would predict that the native molecule is a dimer.
> 100,000, 60,000, and 16,000 are obtained (42). The material The main facts emerging from this work are the following.
with the molecular weight of 60,000 was shown to consist of t’wo (a) Each cell surface contains at least 6 to 11 different glycopro-
chains of nearly idnetical size but different charge (named /3’ and tein subunits although most. of the carbohydrate is present in
p”), and the material with the molecular weight’ of 16,000 only one to three subunits. (b) Each cell surface has a
consisted of one chain (named a). Each of the chains migrated unique glgcoproteiu subunit composition, that is, a glance at
as a dist,inct band in electrophoresis at pI-I 3.5 and 10.3 in 8 h< the Schiff-stained pattern identifies the source. (c) In spite of
urea. The material with the molecular weight of >lOO,OOO the uniqueness of the patterns, some identically sized subunits
was assumed to be an aggregate, but it could not be dissociated are found in all three membranes.
by rechromatography in 5.5 x guanidine hydrochloride or in 8 M These facts raise important questions concerning t’he inter-
urea at pH 3.5 or 10.3, where it migrated as a distinct band upon relations of glycoprotein structure and function. Perhaps the
electrophoresis separated from the o( and fi’ and p” chain. The most interesting question is whether different sized glycoprotein
subunits have different biological functions and whether iden-

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results of SDS electrophoresis are in full agreement with this.
Besides a major glycoprotein (apparent molecular weight 55,000) tically sized subunits hare the same function. Since the
which could represent the p’ or fl” subunit or both of them, a functional specificity of glycoproteins probably resides in the
major subunit with a molecular weight of 17,000 (which could carbohydrate sequences, t,he quest,ion could be rephrased sub-
represent cy) was found. The two glycoproteins with molecular stituting carbohydrate sequence for function.
weights of 95,000 and 135,000 could represent the material If subunit size and carbohydrate sequence are correlated, then
claimed t’o be an aggregate (42). Since the band with a molecular the common bands shared by the differeut cell surfaces represent
weight, of 17,000 did not stain with Schiff stain, one could assume the structural counterparts of common function. Membrane
that t,he o( subunit does not contain carbohydrate, whereas /I’ or processes mediated by glycoproteins which are common to most
0” or both of them contain carbohydrate. cell types are histocompatibility antigens and the lectin binding
Glucose Ozidase-No explanation can be given for the be- sites whose surface exposure is related to cell mitosis (4).
havior of glucose oxidase (A. niger). The molecular Tveight was The membranes which we have studied also have surface
reported to be 153,000 (43). The results of SDS electrophoresis, functions unique for their particular cell type. For example,
howerer, suggest that two glycoprotein chains exist with nearly considerable evidence implicates carbohydrates in cell adhesion;
the same molecular weight and about half of the size reported. however, of the three membranes studied only the liver cell
It should be noted that the holoeuzyme contains 2 moles of membrane normally displays cell adhesion in viva.
FAD (46). ,4 characteristic specialization of brush borders is the presence
Although the number of glycoproteins studied is t,oo small to of high amounts of complex sugar-splitting enzymes (48). Re-
generalize, one can conclude that they can be successfully sepa- cently some of these enzymes have been shown to be glycopro-
rated on SDS gels as subunits and that within the rauge we have teins (49). Beyond the question of carbohydrate sequence and
investigated a good estimation of the molecular weight is pos- subunit size, there is the question of the role of the polypeptide
sible. It, should be noted that the combination of ANS stain backbone of a membrane glycoprotein. The major glycoprotein
and Schiff stain after separation in SDS gels seems to be a power- of the human red blood cell ghost seems to consist of a carbo-
ful method for examination of glycoproteins (or glycopeptides) as hydrate-rich part exposed to the environment aud a region
a purity criterion. consisting mostly of apolar amino acids interacting with the
hydrophobic interior of the cell membrane (2,3). These findings
UISCUSSION
support a model first proposed by Morawiecki (50) which pictures
In order for us to compare membrane glycoproteins by SDS the membrane glycoprotein as a long extended rodlike molecule
gel electrophoresis, it was first necessary to show that the Schiff- with a carbohydrate-rich portion in the aqueous environment and
stained patterns represented size separation of glycoprotein sub- a “tail” which is firmly bound to membrane lipid. Although
units rather than of some undefined artifact. Our results show electron microscopy provides evidence that most of the carbo-
that, providing SDS is first removed, Schiff-stained disc gel
patterns do represent size separations of glycoprotein subunits. 2 The scatter is large because the requirements of a linear plot
In the cases of avidin and ceruloplasmin, both oligomeric glyco- of log RF against molecular weight (25) are variably met for each
proteins, we obtain the monomer molecular weights, indicating glycoprotein. Since only the polypeptide backbone of a glyco-
protein binds SDS (47), the free mobility must be a function of
that our solubilization, reduction, and fractionation methods are the carbohydrate mass. It is also unlikely that the dependence
adequate to achieve separation of subunits. High molecular of mass on Stokes radius is constant 6s the carbohydrate content
weight material which we also resolve in ceruloplasmin is also varies. Until the free mobilities and dependence of Stokes radius
seen after complete reduction and subsequent aminoethylation on mass are determined for glycoproteins, the estimation of
molecular weight by SDS gel electrophoresis must be regarded as
and separation in 5.5 hf guanidinc-HCl (42), and its significance empirical. Alt,hough the method is useful and the standard error
is unknown. for a group of glycoproteins can bc determined, the size of the
The correlat’ion which \I-e find between the logarithm of the error for a single glgcoprotein remains undetermined.
 Glycoproteinns of Cell Surfaces Vol. 246, so. 20

hydrate in cell surfaces is on the outside (5l), one wonders how 21. AMES, B. N., AND DUBIX, D. T., J. Uiol. Chem., 235, 76!) (1960).
22. SPIRO, R. G., ill E. F. NEUFELD -4~1) V. GINSBURG (Editors),
the actual arrangement of membrane glycoproteins is in situ.
Methods in enzymoloy!l, T-01. l?III, Academic Press, New
Examination of plasma membrane glycoproteins by Schiff York, ISGG, p. 3.
staining of SDS gels with the use of continuous buffer systems has 23. G~rr, R., AND BERMLV, E., -1rcuZ. Hiochem., 15, 167 (1966).
previously been reported. The staining method of Zacharius 24. JOVIN, T. K., D.INTE,M. Ii., AND CHR.~.MR.~CH, A., M~lfiphasi~
et al. (28) was used by Lenard (52) and Simon, Blumcnfeld, and bu.fer systems outpui, Federal Scientific and Techuicnl
Information, U. D. Department of Commerce, Springfield,
Arias (53). We hare already pointed out the difficulties which Virginia, 1971.
we encountered when a washing procedure for removing SDS 25. NEVILLE, 1). M., JR., J. Biol. C&n., 246, 6328 (1971,.
was omitted. Our pattern of the human red blood cell ghost xl. GLOSSMANN, H., AX\‘I) LUTZ, F., Hoppe-Seyler’s 2. Physiol.
glycoprot,ein is quite diffrrent from the one published by Lenard Chem., 351, 1583 (1970).
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in liver plasma membrane in the 40,000 t,o 50,000 molecular LOCK, J. J., Anal. Bioehenl., 30, 148 (1969).
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391 (1969).
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 Glycoproteins of Cell Surfaces: A COMPARATIVE STUDY OF THREE
DIFFERENT CELL SURFACES OF THE RAT
Hartmut Glossmann and David M. Neville, Jr.
J. Biol. Chem. 1971, 246:6339-6346.

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