Synthesis of fluorescent oligosaccharides for covalent attachment to living cells

ANALYTICAL

BIOCHEMISTRY

170,520-527

(1988)

Synthesis of Fluorescent Oligosaccharides Attachment to Living Cells’ CARL

G. GAHMBERG

AND MARTTI

Department of Biochemistry, University ofHelsinki,

for Covalent

TOLVANEN Helsinki, Finland

Received October 7, 1987 Asparagine-linked oligosaccharides were liberated from glycoproteins by hydrazinolysis. The treatment resulted in de-N-acetylation of the amino sugars. After isolation of the oligosaccharides free amino groups were labeled with fluorescein isothiocyanate and remaining amino groups reacetylated. The fluorescent oligosaccharides were used to label living cells. They were converted to hydrazine derivatives and covalently attached to cell surface oligosaccharides, which had been treated with periodate or neuraminidase and galactose oxidase. This enabled the visualization of the attached oligosaccharides at the external aspect of the plasma membrane by fluorescence microscopy. 0 1988 Academic PRSS, Inc. KEY WORDS: fluorescence; glycoproteins; membrane proteins; cell surface; cell biology; carbohydrates.

In mammalian cells most surface proteins are glycoproteins (1,2) with their carbohydrate moieties exposed to the external milieu (3,4). There is increasing evidence that the carbohydrate portions act as receptors for various microbes (5) and macromolecules (6) and are important in cell-cell recognition (7-9). Numerous changes in surface glycoconjugates have been observed during cellular differentiation and after malignant transformation (9- 13). The most common carbohydrate chains in glycoproteins can structurally be divided into high-mannoseand complex-type asparagine-linked (N-glycosidic), and serinejthreonine-linked (O-glycosidic) oligosaccharides (14). They all contain amino sugars in their core positions. The presence of cell surface oligosaccharides can be demonstrated using radioactive labeling techniques. By treatment with periodate or galactose oxidase, which oxidize si’ This research was supported by the Academy of Finland, the Sigrid Jus&lius Foundation, the Emil Aahonen Foundation, and the Magnus Ehmrooth Foundation. OOO3-2697188 $3.00 Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form resewed.

alit acids and galactosyl/N-acetylgalactosaminyl residues, respectively, followed by reduction with tritiated borohydride, cell surface glycoconjugates are labeled (3,4,15). Other possibilities include, for example, the use of lactoperoxidase-catalyzed radioiodination to label cell surface proteins (16) followed by lectin affinity chromatography. For visualization of cell surface sugars, fluorescent or heavy-metal-labeled lectins have gained a large popularity. Another widely applicable method is to use compounds such as fluorescent hydrazides, which specifically react with oxidized surface carbohydrate residues ( 17,18). We recently introduced a method by which almost any mono- or oligosaccharide can be covalently attached to sialic acid or galactose/iV-acetylgalactosamine-containing cell surface glycoconjugates (19). In this method glycosyl hydrazines are attached to oxidized cell surface oligosaccharides. This method should make it possible to study specific functions of such “new” carbohydrates at the cell surface. In this context it became important to be able to follow morphologi520

FLUORESCENT

OLIGOSACCHARIDES

tally the fate of the surface-attached carbohydrates. For this purpose we have now developed a simple method which makes it possible to prepare fluorescent derivatives of hexosamine-containing oligosaccharides. MATERIALS

AND METHODS

Glycoproteins. Human transferrin was obtained from Miles Scientific (Naperville, IL). Ovomucoid, fetuin, and ovalbumin were from Sigma Chemical Co. (St. Louis, MO). a ,-Acid glycoprotein was isolated from human urine as described previously (20). Radioactive labeling of glycoproteins. Transferrin, ovomucoid, fetuin, and al-acid glycoprotein (0.1 mg each) were labeled with 3H in their sialic acid residues by the method of van Lenten and Ashwell (2 1) using oxidation with 1 mM sodium meta-periodate (Merck AG, Darmstadt, FRG) at 0°C (15). Excess NaB[3H]4 was destroyed by acidification with acetic acid, and the labeled glycoproteins were isolated by gel filtration on Sephadex G-25 in HzO. Aliquots were counted for radioactivity and the labeled protein peaks lyophilized. Hyrazinolysis and isolation of oligosaccharides. To 20 mg each of transferrin, ovomucoid, fetuin, and al-acid glycoprotein was added an aliquot of the corresponding 3H-labeled protein. Twenty milligrams of ovalbumin was used without tracer. The glycoproteins were lyophilized in screw-cap glass tubes and dried in a desiccator over P205 for at least 24 h. Anhydrous hydrazine (Sigma) (0.2 ml) was added and the tubes were filled with NZ, tightly capped with Teflon-lined caps, and incubated at 105°C for 16 h (22). This treatment liberates the N-glycosidic oligosaccharides and simultaneously results in de-Wacetylation of the N-acetyl hexosamines and N-acetyl neuraminic acid. After cooling on ice, the tubes were directly lyophilized. The oligosaccharides were isolated by gel filtration using a Bio-Gel P- 10 column ( 1 X 95 cm) made in 0.1 M pyridine-acetic acid, pH 5.0. Aliquots were counted for ra-

521

dioactivity using a toluene/Triton X- 114based scintillation fluid. Labeling of oligosaccharides with jluores‘H-labeled transferrin cein isothiocyanate. oligosaccharides were used to define the optimal conditions for labeling with fluorescein isothiocyanate (FITC).’ The incorporation of FITC with time was determined using oligosaccharides obtained from 2 mg of transfen-in for each experimental point. The oligosaccharides were dissolved in 250 ~1 of 0.25 M sodium carbonate-bicarbonate buffer, pH 9.0, and incubated with 1 mg of FITC for indicated times. After incubation 1 ml of saturated NaHC03 was added and the remaining NH2 groups acetylated with 10 ~1 of acetic anhydride. Then more saturated NaHC03 was added, and the acetylation was repeated twice. The sample was then applied to a Sephadex G-25 column (0.7 X 20 cm) in H20 and the fractions containing radioactivity were pooled. The samples were then applied to another Sephadex G-25 column (1 X 100 cm) in H20, and radioactivity and Ad95 were monitored. An LKB Ultrospec 4050 spectrophotometer and glass cuvettes with a light path of 1 cm were used. The void volume fractions containing sugar were pooled, lyophilized, dissolved in 0.135 M NaCl-0.01 M sodium phosphate, pH 8.0, and the Ad95 and radioactivities were determined. This made it possible to estimate the relative amount of FITC incorporated into oligosaccharides. The effect of the concentration of FITC on the efficiency of labeling was determined using the same amounts of transferrin oligosaccharides in the same buffer as above, but varying the concentration of FITC. The time of incubation was 6 h. The fluorescent oligosaccharides were purified as above, and the A&radioactivity ratios were determined. Synthesis of jluoresceinylated glucosamine. In order to determine the amount of FITC bound to oligosaccharides, we synthe’ Abbreviation nate.

used: FITC, fluorescein isothiocya-

522

GAHMBERG

AND

sized radiolabeled fluoresceinylated glucosamine as a model compound for the estimation of the molar absorption coefficient (E& of the hexosamine-conjugated fluorescein group. [6-3H]Glucosamine hydrochloride (22 Ci/mmol, The Radiochemical Centre, Amersham, UK) was diluted into a specific activity of 3.37 X 10’ cpm/mmol with D&Icosamine hydrochloride (Pluka AG, Buchs, Switzerland). This compound (83 pmol) was incubated with 30 pmol of FITC in 1.2 ml of 0.25 M sodium carbonate-bicarbonate buffer, pH 9.0, overnight at room temperature. The reaction mixture was passed through a Sephadex G-25 column (1 X 27 cm) in 0. I35 M NaCl-0.01 M phosphate buffer, pH 7.4, and the fractions which contained both radioactivity and fluorescence were examined by thin-layer chromatography on silica-gelcoated aluminium sheets (Merck DC-Alufolien, 10 X 10 cm) using 1 -propanol:water (85:15) as solvent. Fluorescent bands were observed under uv light and radioactivity was detected by scraping bands of 5 mm of each lane into scintillation fluid. The fractions containing the product (RJ = 0.77) that were devoid of free glucosamine (RJ-= 0.07) were pooled and lyophilized and passed through a similar column of Sephadex G-25 to remove the last traces of free FITC, and the fractions of this run were examined as above. Now no free FITC (Rf = 0.87) appeared in the peak fractions, but very minor fluorescent impurities (not visible in daylight) remained at RJ= 0.70 and 0.62. Radioactivity could be detected only in the major fluorescent band. These peak fractions were used in the determination of the ~495. Determination of the molar absorptivity of jluoresceinylated glucosamine. The absorbances at 495 nm of eight peak fractions which contained the pure fluoresceinylated glucosamine were determined from dilutions 1:60, 1:120, and 1:180 made into 0.135 M NaCl-0.01 M phosphate buffer, pH 8.0. Two samples of 50 ~1 and two of 100 ~1 from each fraction were put into liquid scintillation

TOLVANEN

counting. Knowing the specific activity of the glucosamine that was used in the synthesis allowed calculation of the molar absorption coefficient (6495) using the formula A = ccl where A = absorbance, E = molar absorption coefficient (M-l cm-‘), c = solute concentration (M) and I = length of light path (cm). However, because fluorescein causes considerable quenching in the counting, all 32 values obtained were overestimates. Therefore, the apparent c495was plotted against the product of the counted volume and the A495of the fraction, and the real e495 could be obtained by extrapolating to zero absorbance. Least-square fitting of the data gave the intercept 7.1 X lo4 M-’ cm-’ (coefficient of correlation = 0.996). Determination of the degreeof substitution of oligosaccharides with FIX. Glucosamine in the isolated fluorescent oligosaccharides was quantitated by gas chromatography of trimethylsilyl derivatives using myo-inositol as an internal standard (23). Fluorescein was quantitated by measuring A4g5at pH 8.0 and assuming the same molar absorptivity as in the case of FITC-conjugated glucosamine. Labeling of KS62 cells with fluorescent oligosaccharides. The fluorescent oligosaccharides (50-200 pg) were lyophilized and dried over P205. Then 0.1 ml of anhydrous hydrazine was added, and the tubes incubated at 0°C for 3 h and lyophilized. This treatment did not release the fluorochrome from the oligosaccharides, which was checked by gel filtration of sample aliquots. K562 erythroleukemic cells (24,25) were grown in RPM1 1640 medium supplemented with 10% fetal calf serum, penicillin, and streptomycin. The cells (50 X 106) were washed three times in 0.135 M NaCl-0.0 1 M sodium phosphate, pH 7.4, and divided into three equal aliquots. One aliquot was oxidized with 2 mM meta-periodate at 0°C (15), another one treated for 15 mitt at 37°C with 25 mU of Vibrio cholerae neuraminidase (Koch-Light, Haverhill, UK) and 10 U of galactose oxidase (Sigma) (3), and the third one left untreated on ice. The cells were washed and

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523

OLIGOSACCHARIDES 6

A P

1 20 c x 5,.

x E VO

0 0

I _--20

30

Fraction

40

50

60

number

20

30

40

Fraction

number

50

60

FIG. 1. Gel filtration of oligosaccharides obtained by hydrazinolysis. ‘H-labeled glycoproteins treated at 105°C with hydrazine were run on Bio-Gel P-10 columns and aliquots counted for radioactivity. Patterns obtained from transferrin (A), ol,-acid glycoprotein (B), fetuin (C), and ovomucoid (D). VO, position of void volume.

then incubated at 0°C for 2 h with the fluorescent oligosaccharylhydrazines in 0.135 M NaCl-0.01 M sodium phosphate, pH 7.0 ( 19). After incubation, the cells were washed with ice-cold buffer and immediately subjected to fluorescence microscopy. A Zeiss epifluorescence microscope equipped with the Zeiss filter set 4877 17 for fluorescein was used. Photomicrographs were taken at 400~ magnification on Kodak Tri-X-Pan 400 ASA black-and-white film using a fixed exposure time of 1 min 50 s for all samples in order to reproduce the varying fluorescence intensities.

The major peak eluted with an apparent molecular weight of about 2500. Similar peaks were obtained from al-acid glycoprotein, fetuin, and ovomucoid (Figs. 1B- 1D). The oligosaccharide peaks were pooled and lyophilized as follows: transfenin, fractions 37-46; al-acid glycoprotein, fractions 33-43; fetuin, fractions 36-46; and ovomucoid, fractions 37-50. Ovalbumin oligosaccharides were pooled from the fractions of estimated molecular weights of 1000-3000.

RESULTS

The isolated oligosaccharides were incubated with FITC and re-Wacetylated, and the preparations were subjected to gel filtration on Sephadex G-25 columns made in water. First, a short column was used to get rid of the bulk of salts and free FITC, and a complete separation was achieved on a 1 X loo-cm column. The fractions were measured for radioactivity and their absorbance at 495 nm was determined. Figure 2 shows a representative experiment using transferrin

Isolation of Oligosaccharides after Hydrazinolysis Hydrazinolysis at 105°C resulted in liberation of de-l\‘-acetylated N-glycosidic oligosaccharides. The inclusion of a small amount of 3H-labeled oligosaccharides made the detection of the oligosaccharides simple. Figure 1A shows a Bio-Gel P- 10 gel filtration pattern of labeled transferrin oligosaccharides.

Labeling of Oligosaccharides with Fluorescein Isothiocyanate

524

GAHMBERG

AND TOLVANEN TABLE 2 THEEFFE~TOFTHECONCENTRATIONOFFITCONITS INCORP~RATIONINTOTRANSFERRIN OLIGOSACCHARIDES Concentration of FITC Owfmlf

0 Fraction

number

FIG. 2. Gel filtration of FITC-labeled 3H-labeled transferrin oligosaccharides. The oligosaccharides were chromatographed on a Sephadex G-25 column (1 X 100 cm) in Hz0 and the radioactivities and Adg5 were measured. The FITC-labeled oligosaccharides eluted in fractions 16-20.

oligosaccharides. The FITC-labeled oligosaccharides eIuted in the void volume, whereas the free FITC eluted much later in tubes 30-40. The efficiency of labeling was estimated from the A&radioactivity ratios at pH 8 as described under Materials and Methods. Table 1 shows the incorporation of FITC into a given amount of oligosaccharide with time. After an initial faster reaction, the rate of incorporation slowed down. The incorporation was strongly dependent on the concentration of FITC. Table 2 shows an almost linear increase in incorporation using 0.2- 10 mg/ml of FITC in an incubation of 6 h. Increasing the FITC concentration above 10 mg/ml resulted in precipitation of the reagent.

TABLE

1

Incubation time (h)

Relative specific labeling with FITC

0 0.5 2 4 6

0 0.46 0.65 0.75 1

0.2 1 4 10

Relative specific labeling with RTC 0 0.12 0.44 I

Degree of Substitution of Oligosaccharide Amino Sugars with FITC In order to obtain the molar absorbtion coefficient (t4& for hexosamine-conjugated fluorescein we synthesized radiolabeled fluoresceinylated glucosamine of known specific activity. We obtained the value 7.1 + 0.2 X lo4 M-’ cm-’ for this compound at pH 8.0, which is very close to that given for free FITC at pH 8.0, 7.0 X lo4 M-’ cm-’ (Fluka Reagent Catalog). Assuming that the extinction coefficient would be the same in the oligosaccharide derivatives, we calculated that in our fluorescent oligosaccharide preparations, synthesized using 10 mg/ml FITC and an incubation time of 6 h, the degree of substitution of amino sugar residues with fluorescein is 4-22s (Table 3). (No attempt was made to determine whether glucosamine or sialic acid residues were more heavily labeled.) The results correspond roughly to two fluorescein groups/oligosaccharide from ovomucoid, one fluorescein group/oIigosaccharide from q-acid glycoprotein or fetuin, one fluorescein group/two oligosaccharides from ovalbumin, and one fluorescein group/four oligosaccharides from transfetrin.

Attachment of Fluorescent Oligosaccharides to Living Cells The FITC-labeled oligosaccharides were treated with an excess of anhydrous hydra-

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525

OLIGOSACCHARIDES TABLE

3

WITH FITC OFAMINOSUGARRESIDUESINOLIGOSACCHARIDE

DEGREEOFSUBSTITUTION

PREPARATIONS

FROM

VARIOUS

Source of oligosaccharide

Concentration of amino sugar residues (mM)

q-Acid glycoprotein Fetuin Ovalbumin Ovomucoid Transferrin

3.2 1.3 0.25 1.4 3.1

zine at 0°C. This substitutes the aldehyde groups with hydrazine, without affecting the rest of the oligosaccharides (19). K562 cells were treated either with sodium meta-periodate or neuraminidase and galactose oxidase to generate aldehyde groups at the cell surface and subsequently labeled with the fluorescent oligosaccharylhydrazines. Figures 3A and 3B show that either treatment resulted in an efficient labeling of the cells with the transferrin-derived FITC-labeled oligosaccharides. Nonspecific adsorption of the fluoresceinylated oligosaccharylhydrazines into control cells, which had not been oxidized, was minimal (Figs. 3C and 3D). Similar results were obtained with the oligosaccharides from al-acid glycoprotein, fetuin, ovomucoid, and ovalbumin. DISCUSSION

It is obviously important to determine the two-dimensional location of cell surface glycoconjugates. A lot of work has been done on their visualization using fluorescent ligands, e.g., labeled lectins or carbohydrate-specific antibodies. Another approach has been to oxidize cell surface glycoconjugates with periodate or galactose oxidase for labeling with fluorescent hydrazides or, for example, biotiny1 hydrazide followed by fluorescent avidin or streptavidin (17,26-28). Our approach is different. We want to introduce structurally defined oligosaccharides

GLYCOPROTEINS Concentration fluorescein groups ow 0.28 0.21 0.054 0.30 0.11

of Percentage substitution

of

9 16 22 21 11

into cell surface glycoconjugates by elongating preexisting sugar chains. The oligosaccharylhydrazine method changes the antigenie composition of the cell surface (19), and it may become possible to study the role of carbohydrates in various functional activities attributed to cell surface glycoconjugates. For this purpose it became important to synthesize fluorescent oligosaccharides, so that the fate of the attached carbohydrates could be followed. In preliminary attempts to make fluorescent oligosaccharides, we used periodate-oxidized glycoproteins, incubated these with FITC-hydrazine or dansyl hydrazide to label the aldehyde groups, and reduced the resulting Schiff bases with NaBHe (29). We then cleaved the fluorescent oligosaccharides using endo+N-acetylglucosaminidase F (30). Although the oligosaccharides were liberated, the treatment resulted in breakage of the fluorochrome-NHNH-oligosaccharide bonds. In addition, this method would have had limited applicability because high mannose-type oligosaccharides could not be used, and the use of free reducing oligosacchar-ides as starting material would have resulted in loss of the reducing function. The present method is simple and it can be applied for all protein-bound N-glycosidic oligosaccharides and for any free reducing oligosaccharide which contains amino sugars. It is important to note that strictly anhydrous hydrazine should be used. Dry

526

GAHMBERG

AND TOLVANEN

FIG. 3. Visualization by tluorescence microscopy of tluorescent transferrin oligosaccharides attached to K562 erythroleukemia cells. (A) Pattern observed with periodate-oxidized cells; (B) pattern observed with neuraminidase-galactose ox&se-treated cells; (C) pattern observed with control cells, incubated with fluorescent oligosaccharides without prior oxidation; (D) normal light micrograph of(C). Bar represents 30 pm.

hydrazine is commercially available but it can also be prepared in a well-equipped laboratory (22). Opened vials can be stored in a desiccator over PzOs for weeks. For efficient fluorescein labeling we found the optimal labeling conditions to be incubation with 10 mg/ml FITC in 0.25 M carbonate-bicarbonate buffer, pH 9.0, for 6 h at room temperature. This resulted in substitution of up to 22% of the amino sugar residues with FITC. Instead of FITC, other fluorescent reagents reacting with amino groups, like rhodamine isothiocyanate or 7-amino-4-methylcoumar-in-Zacetic acid N-hydroxysuccinimide ester (3 l), should be possible to use, thus enabling double or triple labeling of cells with different fluorescent oligosaccharides. Fluorescent oligosaccharides certainly have other applications in addition to the one described above. They could be used as ligands for lectins, as substrates for glycosi-

dases or glycosyl transferases, as markers in chromatography, and perhaps as antigens in highly sensitive fluorescence-based immunological assays replacing radioactive antigens. It is possible that fluorescent oligosaccharides could be bound by endogenous cellular lectins (32), and this may also form an interesting line of study. ACKNOWLEDGMENTS We thank Aili Grundstrom and Yvonne Heiniht for expert assistance and Dr. Akira Kobata (Tokyo) for useful advice on the hydrazinolysis reaction.

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OLIGOSACCHARIDES

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