3 Department of Pharmacology and Molecular Sciences, The Johns Hopkins School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205; 4 Instituto de Microbiologia Prof. Paulo de Goes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; and 5 Glycominds, Ltd., Lod 71291, Israel
Received on September 9, 2003; revised on October 22, 2003; accepted on October 22, 2003
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Abstract |
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Key words: CD4+ / glycomics / hepatocyte / lectins / oligosaccharides
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Introduction |
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Many of the proteincarbohydrate interactions that occur at cell surfaces are intrinsically multivalent (Lee and Lee, 2000). Arrays of lectins (or carbohydrates) on one cell surface interact like Velcro with complementary arrays of carbohydrates on an apposing surface. The best characterized example of the kinetic role of multivalency is binding by the hepatic asialoglycoprotein receptor (Lee et al., 1983
). On the intact hepatocyte surface, the site affinity of this lectin for lactose is 300 µM, whereas its affinity for a trivalent oligosaccharide with appropriately spaced galactose termini is 7 nM, nearly five orders of magnitude higher. Similarly, carbohydratecarbohydrate binding may have even lower site affinity, requiring highly multivalent interactions for function (Pincet et al., 2001
). The off rate of a monovalent carbohydrate-mediated interaction may be so rapid that solid-phase binding to immobilized glycan arrays, which require a wash step to remove unbound protein, may be difficult to detect. This problem has been circumvented to some extent by using multivalent soluble lectins or by generating multivalency using chimeras or secondary binding proteins.
In nature, multivalency is often generated by lectin expression on cell surfaces, where lectin molecules self-associate or cluster in response to multivalent binding arrays on an apposing surface (Weis and Drickamer, 1996; Weisz and Schnaar, 1991
). Here we report methods that detect specific adhesion of intact cells to covalent carbohydrate microarrays engineered on glass slides. These methods take advantage of the natural multivalency of cell surface carbohydrate binding to extend the applicability of glycan microarrays.
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Results |
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Discussion |
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The particular glass-slide-based glycan arrays used in the current study possess the following important features for cell-adhesion based screening (Schwarz et al., 2003): (1) Simple, well-characterized chemistry is used to covalently link the glycans to the support; (2) a linker separates the glycan and the solid surface conferring access to cells; (3) the glycans are well defined and bound to the linker by known anomeric bonds; (4) the saccharides are bound by their reducing ends and are presented to cells with a well-defined orientation; (5) the array is produced with high well-to-well and batch-to-batch reproducibility; and (6) simple albumin blocking (hepatocytes) or no blocking at all (T-cells) results in low nonspecific binding, giving excellent signal-to-noise ratios.
The results reported here extend the application of glycan arrays to the quantification of carbohydrate-mediated cell adhesion. This extension provides several potential advantages over the study of soluble lectin binding and broadens the applicability of glycan arrays: (1) Full-length lectins are expressed in their physiological environment at the cell surface; (2) carbohydrate specificity can be screened on primary cells without purifying and expressing the relevant lectin(s); (3) relatively few cells are requiredas few as 6,000 cells per spot (1.2 x 106 cells total per 200-spot slide); (4) the effects of physiological and pathological modulation of cells on their carbohydrate binding specificities can be quantitatively determined; (5) the method can be extended to cultured cells engineered to express full-length cell surface lectins (Collins et al., 2000); and (6) both proteincarbohydrate and carbohydratecarbohydrate interactions are amenable to cell-based microarray studies.
In the current studies, we used both preadhesion (1,1'-dioctadecyl-3,3,3,3'-tetramethyl-indocarbocyanine perchlorate [DiIC18]) and postadhesion (propidium iodide) fluorescent staining to label the cells for detection and quantification of adhesion. The advantage of the later procedure is that cells need not be preincubated with fluorescent label, which may alter their adhesive state. We tested CD4+ human T-cells using both methods (data not shown) and found that preincubation with dye affected binding, in that CD4+ T-cells pre-labeled with DilC18 lost their mannose binding but retained their sialyl Lewis x binding.
The loss of mannose/GlcNAc binding by CD4+ T-cells after preincubation may indicate a lectin of low stability, abundance, and/or affinity. Whereas CD4+ T-cells express L-selectin, a lectin capable of binding sialyl Lewis x (Camerini et al., 1989), expression of intrinsic mannose/GlcNAc binding lectins on these cells has not been established. The mannose/GlcNAc-dependent adhesion that was detected, therefore, may represent a novel lectin activity on CD4+ T-cells. However, it may also be due to other leukocytes present as minor contaminants in the affinity-purified CD4+ cell population (
10% of the cell population, see Materials and methods), some of which express mannose/GlcNAc lectins (East and Isacke, 2002
; McGreal and Gasque, 2002
). The lectin identity and specificity of this binding activity were not analyzed further.
The limitations of cell-adhesion microarray studies are primarily technical. Intact cells are not amenable to long-term storage, cannot be exposed to extreme conditions or detergents, and are less compatible with robotic and high-throughput liquid manipulation systems. Unlike proteincarbohydrate binding assays, cell adhesion requires carefully controlled detachment forces (Schnaar, 1994). As in our previous glycan cell adhesion assays (Collins et al., 2000
; Schnaar et al., 1989
; Swank-Hill et al., 1987
; Yang et al., 1996
), centrifugal force was used for this purpose, requiring the fabrication of acrylic centrifuge carriers so that the cells on the adhesive surfaces were subjected to a uniform detachment force in a sealed, fluid-filled compartment. Although these limitations present a challenge to the application of cell-adhesion based glycan microarray studies, they are offset by the advantages for specific applications. Low numbers of cells required, low background adhesion, reproducibility, and highly specific glycan-dependent cell adhesion were demonstrated using this approach, establishing that intact cells can be used to probe the specificity of carbohydrate-based cell adhesion in a robust microarray format.
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Materials and methods |
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Chicken hepatocytes
Chicken hepatocytes were prepared by collagenase perfusion of juvenile chicken livers as described previously (Obrink et al., 1977). The resulting cell populations were predominantly single cells and >85% viable by trypan blue exclusion. Cell suspensions were maintained at
4°C until use.
An aliquot containing 5 x 106 cells was placed in a 1.5-mL microcentrifuge tube, the cells were collected by centrifugation (15 s, 3000 x g), washed once with 0.53 mM EDTA in calcium-magnesium-free Dulbecco's phosphate buffered saline (PBS), and resuspended in 1 mL of the same solution. After counting, the cells were centrifuged as before and then resuspended at a final concentration of 5 x 106 cells/mL in HEPES buffered Dulbecco's minimal essential medium (DMEM-HEPES) containing 3 µM DiIC18 (Molecular Probes, Eugene, OR). After 1 h at 37°C in the dark with constant gentle agitation, the labeled cells were washed three times with DMEM-HEPES and resuspended at 5 x 106 cells/mL in the same medium supplemented with BSA (5 mg/mL or 10 mg/mL as indicated). In some experiments, cells were suspended in medium containing 25 mM Gal or 25 mM GlcNAc (as indicated) immediately prior to use.
Human CD4+ T-cells
Ten milliliters of peripheral blood were collected from each of seven healthy volunteers into two 10-ml EDTA-Vaccutainers. The blood was incubated for 4 h at ambient temperature. Immediately after incubation, the blood samples were centrifuged (230 x g; 10 min); after the plasma was removed, the upper 2 mL of buffy coat were transferred to a fresh 15-ml test tube. CD4+ cells were then negatively enriched using RosetteSep CD4+ enrichment kit (StemCell Technologies), a procedure that yields 90% CD4+ cells with the remaining 10% representing the other leukocytes in the starting population. An aliquot of cells were diluted 1:10 in Türk solution (0.01% crystal violet and 3% glacial acetic acid) and counted. Cells were further diluted to a density of 5 x 106 cells/ml in RPMI-1640 medium containing 2% fetal bovine serum and incubated overnight in a humidified incubator (37°C, 5% CO2, 95% humidity).
Chicken hepatocyte adhesion to glycan microarrays
Prior to use, glycan array slides were washed with distilled water and air-dried. To reduce nonspecific cell adhesion, an aliquot (1.3 µL) of Dulbecco's PBS containing either 5 mg/mL or 10 mg/mL BSA was placed on each spot, and the slide was incubated in a humid atmosphere (a closed Petri dish with moist filter paper) for 2 h at 37°C. Blocking solution was removed by aspiration and replaced with an aliquot (1.3 µL) of cell suspension (5 x 106 cells/mL). After 30 min at 37°C in a humid atmosphere to allow adhesion to proceed, the slides were gently immersed in a vat of PBS, inverted onto an immersed custom Plexiglas centrifuge carrier (Figure 7), which was then sealed to exclude air. The carrier containing the inverted slides was centrifuged at 800 x g at ambient temperature for 20 min to remove nonadherent cells. The carrier was reimmersed in PBS; the slide was removed while immersed, righted, lifted out of the PBS, and placed gently in a Petri dish containing 2% paraformaldehyde in PBS for 30 min at ambient temperature. The slides were washed by transferring to a Petri dish filled with PBS, sealed under a coverslip, and stored in the dark at 4°C.
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Human CD4+ T-cell adhesion to glycan microarrays
Prior to use, the glycan arrays slides were washed with distilled water and air-dried. The adhesion assay was initiated by applying 1.2 µL human CD4+ T-cell suspension (5 x 106 cells/ml) on each well on the slide; cells from each individual were spotted on replicate slides. After an incubation of 60 min in a humidified incubator (37°C, 5% CO2, 95% humidity) the slides were gently immersed in a vat of PBS, inverted onto an immersed custom acrylic centrifuge carrier (Figure 7), which was then sealed to exclude air. The carrier containing the inverted slides was centrifuged at 50 x g at ambient temperature for 2 min to remove nonadherent cells. The carrier was reimmersed in PBS; the slides gently removed while immersed, then placed in a slide rack. The cells were then fixed to the slides by immersion in PBS/3.7% formaldehyde for 60 min at ambient temperature. The slides were then washed three times in distilled water and air-dried. The bound cells were fluorescently labeled postfixation by incubation of the slides in propidium iodide solution (50 mg/mL in PBS) for 15 min. The slides were then washed three times in distilled water and airdried in the dark.
Fluorescent images of the glycan array slides were captured using an Affymetrix 428 laser scanner (Affymetrix, Santa Clara, CA) using an excitation wavelength of 522 nm and an emission wavelength of 665 nm; fluorescent pixel density was quantified using OptiQuant image analysis software (Packard, Boston, MA).
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Acknowledgements |
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Footnotes |
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2 To whom correspondence should be addressed; e-mail: schnaar{at}jhu.edu
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Abbreviations |
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References |
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---|
Bidlingmaier, S. and Snyder, M. (2002) Carbohydrate analysis prepares to enter the "Omics" era. Chem. Biol., 9, 400401.[CrossRef][ISI][Medline]
Burrows, L., Iobst, S.T., and Drickamer, K. (1997) Selective binding of N-acetylglucosamine to the chicken hepatic lectin. Biochem. J., 324, 673680.[ISI][Medline]
Camerini, D., James, S.P., Stamenkovic, I., and Seed, B. (1989) Leu-8/TQ1 is the human equivalent of the Mel-14 lymph node homing receptor. Nature, 342, 7882.[CrossRef][ISI][Medline]
Collins, B.E., Yang, L.J.S., and Schnaar, R.L. (2000) Lectin-mediated cell adhesion to immobilized glycosphingolipids. Methods Enzymol., 312, 438446.[ISI][Medline]
Crocker, P.R. and Varki, A. (2001) Siglecs in the immune system. Immunology, 103, 137145.[CrossRef][ISI][Medline]
Drickamer, K. (1981) Complete amino acid sequence of a membrane receptor for glycoproteins. J. Biol. Chem., 256, 58275839.
Drickamer, K. and Taylor, M.E. (2002) Glycan arrays for functional glycomics. Genome Biol., 3, REVIEWS 1034.
Dwek, R.A. (1996) Glycobiology: Toward understanding the function of sugars. Chem. Rev., 96, 683720.[CrossRef][ISI][Medline]
East, L. and Isacke, C.M. (2002) The mannose receptor family. Biochim. Biophys. Acta, 1572, 364386.[ISI][Medline]
Fazio, F., Bryan, M.C., Blixt, O., Paulson, J.C., and Wong, C.H. (2002) Synthesis of sugar arrays in microtiter plate. J. Am. Chem. Soc., 124, 1439714402.[CrossRef][ISI][Medline]
Flitsch, S.L. and Ulijn, R.V. (2003) Sugars tied to the spot. Nature, 421, 219220.[CrossRef][ISI][Medline]
Fukui, S., Feizi, T., Galustian, C., Lawson, A.M., and Chai, W. (2002) Oligosaccharide microarrays for high-throughput detection and specificity assignments of carbohydrate-protein interactions. Nat. Biotechnol., 20, 10111017.[CrossRef][ISI][Medline]
Hirabayashi, J. (2003) Oligosaccharide microarrays for glycomics. Trends Biotechnol., 21, 141143.[CrossRef][ISI][Medline]
Houseman, B.T. and Mrksich, M. (2002) Carbohydrate arrays for the evaluation of protein binding and enzymatic modification. Chem. Biol., 9, 443454.[CrossRef][ISI][Medline]
Iwabuchi, K., Yamamura, S., Prinetti, A., Handa, K., and Hakomori, S. (1998) GM3-enriched microdomain involved in cell adhesion and signal transduction through carbohydrate-carbohydrate interaction in mouse melanoma B16 cells. J. Biol. Chem., 273, 91309138.
Kiessling, L.L. and Cairo, C.W. (2002) Hitting the sweet spot. Nat. Biotechnol., 20, 234235.[CrossRef][ISI][Medline]
Lee, R.T. and Lee, Y.C. (2000) Affinity enhancement by multivalent lectin-carbohydrate interaction. Glycoconj. J., 17, 543551.[CrossRef][ISI][Medline]
Lee, Y.C., Townsend, R.R., Hardy, M.R., Lonngren, J., Arnarp, J., Haraldsson, M., and Lonn, H. (1983) Binding of synthetic oligosaccharides to the hepatic Gal/GalNAc lectin. J. Biol. Chem., 258, 199202.
Love, K.R. and Seeberger, P.H. (2002) Carbohydrate arrays as tools for glycomics. Angew. Chem. Int. Ed Engl., 41, 35836, 3513.
McGreal, E. and Gasque, P. (2002) Structure-function studies of the receptors for complement C1q. Biochem. Soc. Trans., 30, 10101014.[ISI][Medline]
Mirzabekov, A. and Kolchinsky, A. (2002) Emerging array-based technologies in proteomics. Curr. Opin. Chem. Biol., 6, 7075.[CrossRef][ISI][Medline]
Obrink, B., Kuhlenschmidt, M.S., and Roseman, S. (1977) Adhesive specificity of juvenile rat and chicken liver cells and membranes. Proc. Natl Acad. Sci. USA, 74, 10771081.[Abstract]
Pincet, F., Le Bouar, T., Zhang, Y., Esnault, J., Mallet, J.M., Perez, E., and Sinay, P. (2001) Ultraweak sugar-sugar interactions for transient cell adhesion. Biophys. J., 80, 13541358.
Schena, M., Shalon, D., Davis, R.W., and Brown, P.O. (1995) Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science, 270, 467470.[Abstract]
Schnaar, R.L. (1994) Immobilized glycoconjugates for cell recognition studies. In Lee, Y.C. and Lee, R.T. (Eds.), Neoglycoconjugates: preparation and application. Academic Press, San Diego, pp. 425443.
Schnaar, R.L., Brandley, B.K., Needham, L.K., Swank-Hill, P., and Blackburn, C.C. (1989) Adhesion of eukaryotic cells to immobilized carbohydrates. Methods Enzymol., 179, 542558.[ISI][Medline]
Schwarz, M., Spector, L., Gargir, A., Shtevi, A., Gortler, M., Altstock, R.T., Dukler, A.A., and Dotan, N. (2003) A new kind of carbohydrate array, its use for profiling anti-glycan antibodies, and the discovery of a novel human cellulose-binding antibody. Glycobiology, 13, 749754.
Swank-Hill, P., Needham, L.K., and Schnaar, R.L. (1987) Carbohydrate-specific cell adhesion directly to glycosphingolipids separated on thin-layer chromatography plates. Anal. Biochem., 163, 2735.[ISI][Medline]
Taylor, M.E. and Drickamer, K. (2003) Introduction to glycobiology. Oxford University Press, Oxford. 207 pp.
Vestweber, D. and Blanks, J.E. (1999) Mechanisms that regulate the function of the selectins and their ligands. Physiol Rev., 79, 181213.
Wang, D., Liu, S., Trummer, B.J., Deng, C., and Wang, A. (2002) Carbohydrate microarrays for the recognition of cross-reactive molecular markers of microbes and host cells. Nat. Biotechnol., 20, 275281.[CrossRef][ISI][Medline]
Weis, W.I. and Drickamer, K. (1996) Structural basis of lectin-carbohydrate recognition. Annu. Rev. Biochem., 65, 441473.[CrossRef][ISI][Medline]
Weisz, O.A. and Schnaar, R.L. (1991) Hepatocyte adhesion to carbohydrate-derivatized surfaces: I. Surface topography of the rat hepatic lectin. J. Cell Biol., 115, 485493.[Abstract]
Yang, L.J.S., Zeller, C.B., and Schnaar, R.L. (1996) Detection and isolation of lectin-transfected COS cells based on cell adhesion to immobilized glycosphingolipids. Anal. Biochem., 236, 161167.[CrossRef][ISI][Medline]