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Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
1Correspondence: Massachusetts Institute of Technology, Bldg. 68–480, 77 Massachusetts Ave., Cambridge, MA 02139, USA. E-mail: rdrrosen{at}mit.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: heparin • gel mobility shift assay • in vitro modification • antithrombin III • sulfotransferase
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3)
. For example, HS is critical for the entry of herpes simplex virus type 1 (4)
; HS chain of syndecan-3 is found to play significant role in modulating feeding behavior in mice (5)
; HS modulates the association between fibroblast growth factors (FGFs) and their receptors (FGFRs) (6
7
8)
. The biological significance of HS is also manifested at the whole animal level, where genes involved in HS biosynthesis are deficient (9)
. Mice lacking glucosaminyl N-deacetylase/N-sulfotransferase 1 and HS 2-O sulfotransferase (2-OST) died neonatally (10
, 11)
. Homozygous mutation of one of the HS polymerases, EXT1, led to embryonic lethality due to a failure to gastrulate (12)
. Drosophila with mutations of UDP-D-glucose dehydrogenase or N-deacetylase/N-sulfotransferase (NDST) lost wingless activity as well as FGF and hedgehog signaling pathways (13
14
15)
. Altered expression of heparan sulfate proteoglycan led to human diseases such as chondrodystrophic myotonia (16)
and hereditary bone disorders (17)
.
Catalyzed by HS polymerases, HS is initially synthesized in the Golgi apparatus as a nonsulfated copolymer attached to HS proteoglycan core proteins by sequential addition of D-glucuronic acid alternating with N-acetyl D-glucosamine. This is followed by various modification steps including N-deacetylation and N-sulfation of glucosamine, epimerization of GlcA to L-iduronic acid, 2-O sulfation of uronic acid, and 6-O sulfation and 3-O sulfation of glucosamine. All steps are catalyzed by different enzymes and the process is selective as to the position and number of modifications in a chain, leading to extensive sequence diversity (2
, 18)
. Except for 2-O sulfotransferase (19)
and epimerase (20)
, five isoforms of 3-O sulfotransferase (3-OST) (21)
, three isoforms of 6-O sulfotransferase (6-OST) (22)
, and four isoforms of NDST (23)
have been cloned. In addition, some isoforms possess allelic variants and/or exhibit splicing variants. These isoforms are not redundant, but each has its own substrate specificity, tissue expression pattern, and unique function (2
, 18)
. For example, 3-OST-1 is responsible for the antithrombin III (AT-III) binding sequence and 3-OST-3A is critical for gD binding of herpes simplex virus, although the normal biological function of 3-OST-3A is unknown (4)
. We have expressed most of these modification enzymes, enabling us to perform in vitro modification of heparan sulfate in different combinations.
HS functions by interacting with various proteins. Hundreds of HS binding proteins have been identified and the number is increasing rapidly. These proteins fall into diverse groups, including proteins of the circulatory system, growth factors, receptors, adhesion proteins, enzymes, cytokines, chemokines, protease inhibitors, and virus proteins; in only a few cases have the polysaccharide features and modification enzymes been defined (18
, 24)
. There is little doubt that deciphering these features will help to settle fundamental issues in development, physiology, and behavior processes. As an illustration, we use a novel strategy with the combination of in vitro modification and gel mobility shift assay (GMSA) to reveal the structural features of heparin oligosaccharide, which recognizes and activates AT-III.
GMSA has been used to study protein–DNA interaction and shown to be successful in identifying specific DNA sequences and the cognate proteins (25)
, but to the best of our knowledge this is the first time that GMSA has been systematically used to study the interaction between protein and heparin. Unlike DNA, in which the sequence diversity is inherited from parental DNA, heparin or HS sequence diversity is generated by incomplete enzymatic modifications in the Golgi apparatus. This led us to the idea of changing or building the sequences of heparin or HS by in vitro modification. The effect of modification could then be revealed by GMSA.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Completely desulfated and N-resulfated heparin sulfate (DSNS) was from Seikagaku America (Falmouth, MA). APS kinase was a generous gift from Dr. Irvin Segel (University of California-Davis). Other biochemicals were from Sigma (St. Louis, MO).
Preparation of [35S]PAPS
For each reaction, the following materials were mixed (27)
: 40 µl of 50 mM MgATP, 20 µl of 100 mM MgCl2, 10 µl of Na235SO4 (10 mCi from ICN), 25 µl of 160 mM phosphoenol pyruvate (PEP), 1 µl of 700 units/ml pyruvate kinase, 1 µl of 250 units/ml inorganic pyrophosphatase, 2 µl of 15 units/ml APS kinase, 2 µl of 2 units/ml ATP sulfurylase, 69 µl of 50 mM Tris, pH 8.0. The final reaction volume was brought to 170 µl by adding water. The reaction was incubated overnight at 30°C. The [35S]PAPS was then purified on a DEAE-Sephacel column. In a 0.8 x 4 cm chromatography column, 0.5 ml bed volume of DEAE-Sephacel was packed and equilibrated with 10 ml of 10 mM triethyl-ammonium bicarbonate buffer, pH 8.0. After loading the reaction mixture, the column was washed with 5 ml of 200 mM TEA buffer, pH 8.0. The bound [35S]PAPS was eluted with 2.5 ml of 400 mM TEA buffer, pH 8.0. The eluant was lyophilized and resuspended in 170 µl of 1 mM Tris buffer, pH 8.0. The concentration was measured at an absorbance of 260 nm.
Radiolabeling and in vitro modification of oligosaccharide
The labeling 2x buffer contains 50 mM MES (pH 7.0), 1% (W/V) triton X-100, 5 mM MgCl2, 5 mM MnCl2, 2.5 mM CaCl2, 0.075 mg/ml protamine chloride, 1.5 mg/ml BSA. For a 25 µl reaction, the following were assembled: 2 µg of substrate (heparan sulfate or heparin), 12.5 µl of 2x buffer, 70 ng of the expressed sulfotransferase, 2 µl [35S]PAPS (
1.0x107 cpm), and the appropriate amount of water. The reaction was incubated at 37°C for 20 min, then stopped by heating to 75°C for 3 min. The reaction was centrifuged at 10,000 g for 3 min and the supernatant was used for GMSA.
Gel mobility shift assay
The heparin–protein binding buffer contained 12% glycerol, 20 mM Tris-HCl (pH 7.9), 100 mM KCl, 1 mM EDTA, and 1 mM DTT. For a typical 20 µl binding reaction, 10 ng of oligosaccharide (10,000 cpm) was mixed with an appropriate amount of AT-III in the binding buffer. The reaction was incubated at room temperature (23°C) for 20 min. Multiple reactions were usually carried out at the same time. Half of the reaction (10 µl) was then applied to a 4.5% native polyacrylamide gel (with 0.1% of bis-acrylamide). The gel buffer was 10 mM Tris (pH 7.4) and 1 mM EDTA, and the electrophoresis buffer was 40 mM Tris (pH 8.0), 40 mM acetic acid, 1 mM EDTA. The gel was run at 6 volts/cm for 1–2 h with an SE 250 Mighty Small II gel apparatus (Hoefer Scientific Instruments, San Francisco). After electrophoresis, the gel was transferred to 3 MM paper and dried under vacuum.
Autoradiograph and gel analysis
The dried gel was autoradiographed by a PhosphorImager 445SI (Molecular Dynamics, Sunnyvale, CA). The image was analyzed with NIH Image 1.60 and the band intensities were evaluated. The concentrations of bound and free oligosaccharides in the binding reaction were derived from the ratio of the respective bands. The dissociation constant was then calculated based on concentrations of free and bound oligosaccharides and protein.
Baculovirus expression and purification of various sulfotransferases
All sulfotransferases were cloned and expressed in COS or Baculovirus as described previously (28
29
30)
.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. [35SO4] was then added to the pentasaccharide by 3-OST-1 and/or 6-OST-1. Products with [35SO4] at the critical 3-O position only, at the critical 6-O position only, and at both positions were named 3S, 6S, and 3S6S, respectively (Fig. 1
A).
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The modified pentasaccharides were next subjected to GMSA. As expected, 6S, which lacked the critical 3-O sulfation, did not bind to AT-III and thus was not retarded by AT-III, but the 3S6S could bind to AT-III and be shifted almost completely. On the other hand, 3S, which lacked the critical 6-O sulfation, was very slightly shifted by AT-III (Fig. 1B
). After densitometry analysis, the binding affinity of 3S6S to AT-III was estimated to be 80-fold that of 3S. 3S, 6S, and 3S6S were observed to have different mobilities, indicating that modification affected the charge density and molecular structure of the oligosaccharide. This experiment proved that 3-O and 6-O sulfates work in a thermodynamically linked fashion to cooperatively increase the binding affinity for AT-III and validated the gel shift assay for studying protein–HS interaction.
Measurement of affinity between AT-III and the fully sulfated pentasaccharide
The affinity to proteins is essential for functions of HS. GMSA was applied to measuring the binding affinity between DNA and transcription factors (25
, 32)
. We measured the affinity between 3S6S and AT-III using GMSA. In this experiment, the same amount of pentasaccharide (120 ng, with a specific activity of 3.3x106 cpm/µg) but an increasing amount of AT-III were added to each binding reaction. The binding was visible at level of 50 ng of AT-III (<1 pmol) (Fig. 2
A). The radiogram was analyzed with NIH Image 1.60 program and the peak values were collected (Fig. 2B
). The concentrations of free and bound AT-III were then derived. The dissociation constant Kd was derived with Scatchard plot (Fig. 2C
) to be 0.16 µM, which is comparable to previous measurement of 0.1 µM (33)
. Affinity measurement and structural selectivity evaluation of heparin by electrophoresis was introduced in a pioneering investigation by Lee and Lander (34)
, whereas our strategy allows for studying the functional features on HS.
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Minimum length and the length effect of heparin for AT-III binding
Information on minimal binding sequences on HS is vital to understanding the rules of HS–protein interaction and design of HS mimics that can target proteins in human diseases. We measured the minimal binding length for AT-III. To do this, an oligosaccharide ladder from tetrasaccharide to octadecasaccharide was radiolabeled with 3-OST-1 (Fig. 3
A). The probes were incubated with same amount of AT-III and separated on a 4.5% native PAGE gel. The binding between the oligosaccharides and AT-III was shown as the shifted bands. Although significant amount of hexasaccharide and larger oligosaccharides could be shifted by AT-III, tetrasaccharide was not shifted (Fig. 3B
). Considering that the pentasaccharide 3S6S bound to AT-III strongly (Fig. 1B
), it was concluded that the minimal length for AT-III binding was a pentasaccharide. It was also noticed that increasing the size of oligosaccharide resulted in more binding, perhaps because the chance for an AT-III binding site in a longer oligosaccharide is higher than in a short one.
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Specific fraction of oligosaccharides binds to AT-III
HS is heterogeneous in nature. When studying the interaction between HS and a protein, it is important to know whether the interaction is sequence specific (35)
. In the case of a specific interaction, only the fraction of HS with right sequences can interact with the protein. Since only a fraction of HS can bind to the protein, the interaction is sequence specific. With GMSA, we can easily determine if this occurs. An octadecasaccharide (a chemical analog of HS oligosaccharide) was radiolabeled with 3-OST-1 and/or 6-OST-1, and the modified oligosaccharides were subjected to GMSA (Fig. 4
A–C). The gel was then analyzed with densitometry. In each case, the amount of shifted band increased with AT-III, and this increment approached a plateau value. For 6-OST-1 modification, 25% of the oligosaccharides could bind to AT-III. For 3-OST-1 modification, 40% could bind to AT-III. For the double modification, 50% could bind to AT-III (Fig. 4D
). The small additional increments at a high level of protein were likely caused by nonspecific binding. This experiment proved that the interaction between heparin oligosaccharide and AT-III is sequence specific.
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In vitro reconstitution of AT-III binding sites
Since the most critical modifications for AT-III binding are 3-O and 6-O sulfations, we wondered whether we could reconstitute AT-III binding sites on completely desulfated and N-resulfated heparin sulfate (DSNS) by addition of 3-O and 6-O sulfates to the chain. DSNS was first modified with different combinations of sulfotransferases. The extent of incorporation of [35S]PAPS differed from enzyme to enzyme, but in each binding assay, roughly the same amount of radioactivity was used. When the chain was modified by 6-OST-1 alone, the chain could not bind to AT-III at all. Only a slight amount of 2-OST-labeled chain could bind to AT-III. The 3-OST-1-labeled chain obviously bound to AT-III. When the chain was modified by 6-OST-1 and 3-OST-1, it strongly bound to AT-III; further addition of 2-OST did not change the binding significantly, which was consistent with the earlier finding that 3-O and 6-O sulfation was more critical than 2-O sulfation (33)
(Fig. 5
A).
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Since there are multiple isoforms for 3-O and 6-O sulfotransferases, we asked whether those isoforms could substitute for 3-OST-1 and 6-OST-1 in generating AT-III binding sites. We found that 3-OST-2, 3-OST-3A, and 3-OST-4 could not substitute for 3-OST-1 in making the AT-III binding sites, and 6-OST-2A, 6-OST-2B, and 6-OST-3 could substitute for 6-OST-1 (Fig. 5B
). These experiments showed that 3-O sulfation by 3-OST-1 and 6-O sulfation are the critical modification steps in making AT-III binding sites (28)
and that 6-O sulfation can be carried out by different isoforms. These data were consistent with our previous discovery that 6-OST-1 was a critical modification enzyme in CHO cell for making AT-III binding sites in a somatic mutagenesis study (36)
. More important, this proved it is possible to generate HS libraries and reconstitute protein binding sites on DSNS chain by in vitro modification.
GMSA with other proteins
To demonstrate that our approach could be applied to any HS binding protein, we studied interactions among FGF, FGFR, and heparin oligosaccharides. A heterogeneous tetradecasaccharide sample was radiolabeled with 3-OST-1 and incubated with 250 ng of various recombinant proteins. Except for FGFR2, all bound to the labeled oligosaccharide, as shown by the shifted bands (Fig. 6
A). The position of each shifted band varied from protein to protein, reflecting the differences in conformation, mass, and charge density of the oligosaccharide–protein complexes. FGF2 almost completely shifted the probe, indicating that it may have the least selectivity toward the oligosaccharides (Fig. 6A
). When FGF1 and FGFR1 were combined in the binding assay, two bands appeared. The fast-moving band was FGF1–oligosaccharide complex. The slow-moving band should be a FGF1-FGFR1–oligosaccharide complex, since the amount of the fast-moving band decreased and that of the slow-moving band increased when more FGFR1 was added (Fig. 6B
). This experiment proved that in vitro modification and GMSA could be a general approach for studying heparan or heparin sulfate and protein interaction.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 37)
. How HS interacts with proteins and affects the stability, conformation, concentration, and activity of proteins is a fundamental question in biology. In this paper we have described a rapid, convenient, sensitive, and inexpensive strategy to study heparin–protein interaction by combination of in vitro modification and GMSA. This strategy allowed us to establish critical parameters in HS–protein interaction, generate HS libraries, and reconstitute protein binding sites on completely desulfated and N-resulfated heparin.
Though HS is similar to DNA in some ways, there is neither molecular cloning nor a similar sequencing technology for HS. Most studies of HS involve affinity purification to obtain a ‘homogeneous’ HS population, but several factors make it almost impossible to obtain such a sample: HS is tremendously heterogeneous in sequence and size; proteins may recognize motif structures instead of single defined sequences; the interaction between HS and protein, as it should be, is relatively weak; and the source of HS is usually limited. In addition, most current methods for studying HS–protein interaction involve immobilization or chemical labeling of either component, which may introduce more difficulties and artifacts. Recently, technologies for sequencing polysaccharides have been developed (38
39
40)
, but with the difficulties mentioned, very few oligosaccharide sequences have been obtained so far. For most cases, perhaps, there are a variety of HS sequences capable of binding to a single protein, and what really matters in the recognition and activation of a protein is the critical functional groups on HS.
Our approach allows the study of the critical groups on heparin or heparan sulfate by avoiding the above problems. In a natural sample of HS or an HS library generated by in vitro modification, only the oligosaccharides bound to proteins will be revealed as shifted bands in GMSA. With this strategy, it should be possible to determine the critical functional groups on any protein binding HS, to establish whether those groups work in a cooperative manner, and reveal the relationship between modification enzymes, functional groups, and biological functions. After determination of the HS structural features recognized by a certain protein, it will be possible to generate oligosaccharides with the same feature by in vitro modification and to use the oligosaccharide as a drug to block biological activities, such as the binding of gD of herpes simplex virus (4)
or Tat of human immunodeficiency virus (41)
to the HS receptors on host cells. Because no homogeneous HS is required and due to the greater sensitivity of GMSA, it will be practical to prepare in vivo labeled HS from tissues of different development stages and types and to determine how HS affects a specific biological process.
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ACKNOWLEDGMENTS |
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Received for publication October 24, 2001.
Revision received December 12, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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