Bioconjugate Chem. 1994, 5,370-372
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TECHNICAL NOTES Biotinylated Hyaluronic Acid: A New Tool for Probing Hyaluronate-Receptor Interactions Tara Pouyani and Glenn D. Prestwich’ Department of Chemistry, University at Stony Brook, Stony Brook, New York 11794-3400. Received November 12, 1993”
Hyaluronic acid (HA) is a linear polysaccharide composed of repeating disaccharide units of D-glucuronic acid (GlcUA) and N-acetyl-D-glucosamine (GlcNAc). Hyaluronate plays an important role in many biological processes as mediated by its interactions with a number of HA-binding proteins (the “hyaladherins”) and with the cell surface HA-receptor, CD44. Studies of hyaluronate-hyaladherin interactions would be greatly facilitated by the availability of molecular probes derived from HA. We recently reported a convenient chemical modification of hyaluronate that introduces multiple pendant amine functionalities onto the HA carboxylate residues. We now report the preparation of biotinylated hyaluronic acid (molecular weight = 1.2 X lo6 Da) as a probe for histochemical and immunochemical characterization of HA-binding proteins. Approximately one-third of the available HA glucuronate residues could be readily biotinylated in high molecular weight HA.
INTRODUCTION Modification of biopolymers with reporter groups has become a powerful research tool in immunology, cell biology, and histochemistry (1). Generally, this strategy involves binding small molecules with specific reporter properties, e.g., fluorophores, antigens, or radioisotopes, to the biopolymer. The targeted biological macromolecule often has primary amino (NHz), thiol (SH), or other pendant functionality that is readily derivatized with group-specific reagents. Hyaluronic acid (HA), usually isolated as sodium hyaluronate, is a biologically important biopolymer (Figure 1)that plays important roles in diverse processes such as cell motility (21, wound repair (31, and cancer metastasis ( 4 ) . The availability of hyaluronate probes would greatly facilitate the study of the interactions of HA with its binding proteins (hyaladherins) and could potentially allow the localization of these proteins. Ideally, one would want to use either HA oligosaccharidesor high molecular weight HA, and one would want precise control of the degree and chemistry of derivatization. A number of research groups have reported the use of biotinylated HA as a probe for HA binding proteins. However, a reliable and versatile method for preparing biotinylated HA probes is currently unavailable ( $ 6 ) . Three methods are currently used. First, biotinylated HA was prepared by modification of a small number of putative free amino groups believed to be present on native hyaluronate (7). A second approach for the preparation of polysaccharide probes used cyanogen bromide activation of the hydroxyl groups. This has the disadvantage that it introduced random modifications along the polymer backbone (8). Third, HA has one “reducing-end” sugar per molecule; this could be reductively coupled to a diamine and used to prepare HA probes. Unfortunately, derivatization of a single reporter group to a M , 1.5 X lo6 Da molecule gave a probe with low sensitivity of detection (9).
* Abstract published in Advance ACS Abstracts, May 1,1994. 1043-1802/94/2905-0370$04.50/0
nu
\
OH
GlcUA
GlcNAc
GlcUA
GlcNAc
Figure 1. Structure of hyaluronic acid (HA) showing two disaccharide repeat units. We recently described a convenient methodology that allows the controlled attachment of a pendant hydrazido moiety to the glucuronic acid residues of the HA backbone via a variable-length spacer (Figure 2) (10-12). We now report extension of this methodology to prepare biotinylated native HA ( M , = 1.2 X lo6 Da) through the reaction of the pendant hydrazido group with sulfo-NHS-biotin. The technique is general and can be applied to HA oligosaccharides in any size range. EXPERIMENTAL PROCEDURES Materials. Sodium hyaluronate (Cristalhyal) was provided by Collaborative Laboratories, Inc. (East Setauket, NY). Adipic dihydrazide and l-ethyl-3-[3(dimethylamino)propyl]carbodiimide (EDC) were purchased from Aldrich Chemical Co. (Milwaukee, WI). Sulfo-NHS-biotin, 2-(4’-hydroxyphenylazo)benzoic acid) (HABA),and avidin were purchased from Pierce Chemical Co. (Rockford, IL). Spectrapor membrane tubing (MWCutoff = 3500 Da) was obtained from Fisher Chemical Co. (Pittsburgh, PA). Preparation of Hydrazido-HA (1). Sodium hyaluronate (200 mg, 0.50 mmol) was dissolved in water such that the concentration of the HA solution was approximately 4 mg/mL. To this mixture was added a 30fold molar excess of adipic dihydrazide (3.5 g, 20 mmol). The pH of the reaction mixture was then adjusted to 4.75 using 0.1 N HCl. To this mixture was added EDC (382 mg, 2.0 mmol) in solid form. The pH of the reaction 0 1994 American Chemical Society
Technical Notes
Bioconjugate Chem., Vol. 5, No. 4, 1994 371 0
< -
II
Probe-NHS pH = 7.0.8.5
NH-NH-Probe
fl Probe = Biotin
Figure 2. General strategy for generating molecular probes of hyaluronate.
mixture was maintained at 4.75 by addition of 0.1 N HC1. The reaction was allowed to proceed for 2 h or until no further rise in pH was observed. The pH of the reaction mixture was then raised to 7.0 by addition of 1N NaOH. For purification, the reaction mixture was transferred to the prewetted dialysis tubing and was dialyzed exhaustively against water. The clear and viscous final mixture was placed on the lyophilizer for 48 h. Preparation of Biotinylated Hyaluronate (3). Hydrazido-HA (1) (11mg, 0.028 mmol) was dissolved in 0.1 M NaHC03 to a concentration of 7 mg/mL. To this solution was added sulfo-NHS-biotin (2) (50 mg, 0.11 mmol) in solid form, and the resulting turbid reaction mixture was stirred for 18h at ambient temperature. The mixture was then diluted 10-fold with water, transferred to pretreated dialysis tubing, and dialyzed exhaustively against water. The biotinylated hyaluronate probe 3 was isolated as a white fiber after lyophilization. The degree of substitution was determined by a displacement assay according to the manufacturer’s protocol (Pierce). Briefly, 900 p L of avidin-HABA reagent was placed in a 1-mL cuvette. The absorbance at 500 nm was recorded. To this mixture was added 100 pL of a 0.25 mg/mL sample of biotin-HA (3) in phosphate-buffered saline. After thorough mixing the absorbance at 500 nm was recorded. These data were used to calculate the degree of substitution using the reported value for the extinction coefficient of biotin (34 pmol/mL). An average value of 0.33 mol of biotin/mol of HA was obtained from three separate measurements on a single preparation. RESULTS
Native hyaluronate was dissolved in water to a final concentration of 4 mg/mL, and adipic dihydrazide was added in 20-50-fold excess. The pH of the reaction mixture was adjusted to 4.75, and the coupling reagent EDC was added to the mixture in solid form. The pH of the reaction was maintained at 4.75 by addition of 0.1 N HC1 and was allowed to proceed for 2 h or until no further increase in pH was observed. The mixture was dialyzed exhaustively against water, and hydrazido-HA 1was isolated as a white fiber after lyophilization. The hydrazido-modified HA 1was then dissolved in 0.1 M NaHC03 at a concentration of 7 mg/mL. Sulfo-NHSbiotin (2) was added to this clear solution in solid form. After the reaction mixture was stirred for 18 h at ambient temperature, the mixture was diluted 10-fold with water and dialyzed exhaustively against water. The biotinylatedHA probe 3 (Scheme 1)was isolated in quantitative yield after lyophilization. The degree of substitution was determined by a spectrophotometric displacement assay to be 0.33 mol biotin/mol of HA. DISCUSSION
The major objective of this paper is to present a convenient and reproducible method for the preparation of a biotinylated-HA probe. Methodology that we had
Scheme 1. Preparation of Biotinylated Hyaluronic Acid
1
2 0.1 M NaHC03 pH = 8.50
previously developed for HA oligosaccharides of defined length was extended to high molecular weight hyaluronate (1.2 X lo6Da) to provide hydrazido-HA (1). The pendant hydrazido group represents a highly versatile functionality that can react with a number of commercially-available amine-specific reagents with reporter functions. Reaction of the hydrazido-functionalized HA 1 with sulfo-NHSbiotin (2) in 0.1 M NaHC03 (pH = 8.50) resulted in the formation of the HA-biotin complex (3). We have described a coiivenient, simple, and reproducible method for preparing biotinylated HA (3). This technique has five important advantages over earlier methods. First, all reaction components are water-soluble, eliminating the need for the use of cosolvents. Second, all reactions are conducted between pH 4.75 and 8.5; these mild reaction conditions prevent the degradation of HA. Third, the presence of the six-carbon spacer of the adipate moiety further ensures the availability of biotin to the binding site of avidin, affording increased sensitivity of detection. Fourth, the degree of substitution can be readily varied by keeping a large excess of dihydrazide relative to HA and varying the proportion of EDC to HA during the initial coupling reaction. Finally, this methodology allows the introduction of high levels of biotin since there are multiple attachment sites on the HA backbone. Up to one-third of the glucuronate moieties could be derivatized using this method. Alternatively, reducing the modification of the HA would provide a lower loading of biotin groups. ACKNOWLEDGMENT
We thank the Center for Biotechnology funded through the New York State Science and Technology Foundation for financial support of this project. Sodium hyaluronate (Cristalhyal) was kindly provided by Dr. James Hayward of Collaborative Laboratories, Inc. LITERATURE CITED (1) Brinkley, M. (1992) Bioconjugate Chem. 3, 2-13. (2) Banerjee, S. D., and Toole, B. P. (1992) J. Cell Biol. 119, 643-652. (3) LeBoeuf, R. D., Raja, R. H., Fullert, G. M., and Wiegel, P. H. (1986) J . Biol. Chem. 261, 12586-12592. (4) Turley, E. (1984) Cancer Met. Rev. 3, 325-339.
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(5) Hardwick, C., Hoare, K., Owens, R., Hohn, H. P., Hook, M., Moore, D., Cripps, V., Austen, L., Nance, D. M., and Turley, E. A. (1992)J: Cell Biol. 117, 1343-1350. (6) Yang,B.,Zhang, L., and Turley, E. A. (1993)J.Biol. Chem. 268,8617-8623. (7) HUdinghm, T. E.,and Fosang, A. J. (1992)FAsEB J. 6, 861-870. (8) Glabe, C. G., Harty, P. K., and Rosen, S. D. (1983)Anal. Biochem. 130, 287-294.
Pouyani and Prestwich (9) Raja, R. H., LeBoeuf, R. D., Stone, G. W., and Weigel, P. H. (1984)Anal. Biochem. 139, 168-177. (10) Pouyani, T.,and Prestwich, G. D. (1994)Bioconjugate Chem., in press. (11) PouYani~T.,Hubison, G. s.9and Prestwich, G. D.(1993)J . Am. Chem. SOC.,submitted. (12) Pouyani,T.,andPrestwich, G.D. Functionalizedderivatives of hyaluronic acid. Patent Appl. Nov, 1993.
