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ISSN 0582-9879                 ACTA BIOCHIMICA et BIOPHYSICA SINICA 2001, 33(2): 153-157                   CN 31-1300/Q

 

Identification by Site-directed Mutagenesis of Amino Acid Residues Flanking RGD Motifs of Snake Venom Disintegrins for Their Structure and Function

XU Cun-Shuan*, RAHMAN Salman1

(Life Science Institute, Henan Normal University, Xinxiang 453002, China; 1Hamophilia Research Center, University of London, London SE1 7EH, United Kingdom )

 

Abstract  In order to demonstrate that the amino acid residues flanking the RGD sequence were important for inhibiting the ADP-induced platelet aggregation, we analyzed the role of the amino acid residues in the domain preceding the RGD loop on the activity of disintegrins. Our approach was to develop hybrids between the disintegrins kistrin and elegantin targeting residues in this domain and within the RGD loop. The basic sequence within elegantin KKKR44T45I46/A50RGDN54P55 was changed by mutagensis to SKAG44T45I46/P50 RGDM54P55 and to SKAG44I46/P50RGDM54P55, thereby resembling the corresponding S39RAGT43/P50RGDM52P53 sequence in kistrin. This changed KKKR44T45I46/A50RGDN54P55→SKAG44T45I46/P50RGDM54P55 dramatically reduced the activity of elegantin as an inhibitor of platelet aggregation. In contrast, deletion of T45 (KKKR44T45I46/A50RGDN54P55→SKAG44T45I46/P50RGDM54P55) increased activity of elegantin as an inhibitor platelet aggregation. It was further shown that their electrophoresis properties were very different. These data highlight the importance of the domain encompassing residues 39-45 and the amimo acid residues flanking the RGD sequence on disintegrin structure-function.

Key words  site-directed mutagenesis; integrin; disintegrin; RGD motif; viper venom

 

Cell-cell and cell-matrix reactions are mediated by cytoadhesive receptors expressed on the cell surface. Among these receptors, named as integrins, comprise a superfamily of transmembrane heterodimeric molecules and includes the platelet membrane glycoprotein (GP) aIibb3, which plays a role in the platelet aggregation[1]. Integrin function provides a two-way flow of information between the cell and its external environment manifested by the capacity of integrin complexes to regulate both the assembly of an extracellular matrix and cellular behaviour in response to ligand engagement[2]. Two processes are fundamental to integrin function: affinity modulation, which physiologically is thought to be predominantly controlled by a process termed “inside-out” signal transduction and ligand recognition, which is dependent upon the structure of the extracellular domain of the integrin and its cognate ligand[3].

Ligand binding is a complex process involving changes in the conformation of both the receptor and the ligand. Despite this complexity, some basic principles underlying the recognition between integrins and their ligands have been elucidated by the identification of short peptide motifs within extracelluar matrix proteins responsible for integrin binding activity. Many physiological and non-physiological integrin ligands utilize RGD, LDV, MGD or related sequence as key structural components of their receptor binding domain[4,5]. NMR and X-ray crystallography studies have demonstrated that these integrin recognition motifs are generally maintained within solvent exposed b-loop structures that are located in a variety of different protein modules of distinct three-dimensional structure[5－11].

Disintegrins are a family of platelet aggregation inhibitors derived from viper venoms. Previous studies on disintegrins have implicated important functions for the amino acid residues immediately flanking the RGD sequence in regulating the specificity of the integrin-ligand interaction[12, 13]. For example, Scarborough and his co-workers observed that disintegrins with a RGDW motif display higher activity for the integrin aIibb3, whereas disintegrins with an RGDN sequence interact more strongly with avb3 and a5b1 integrins[12]. We reported that structurally unrelated venom proteins kistrin and dendroaspin harboring identical RGD flanking residues (PRGDMP) showed analogous inhibitory properties in platelet aggregation and adhesion assays and could compete with one another in a simple linear competitive manner for binding to the aIibb3 complex[13, 14]. In contrast, elegantin (ARGDNP), although structurally related to kistrin, showed distinct inhibitory properties in platelet aggregation and adhesion assays and was inhibited by kistrin (and dendroaspin) in a non-competitive manner. Definitive evidence for the regulatory function of the RGD flanking residues was obtained by introduction of both single and double amino acid substitutions around the RGD sequence in dendroaspin and elegantin with altered functional consequences on both structural templates.

In the present work we have analyzed the functional role of the domain preceding the RGD loop and the amino acid residues close flanking the RGD sequence by generating hybrids between the two disintegrins kistrin and elegantin. We substitutes the residues KKKR44T45I46/RGDN54P55→SKAG44T45I46/RGDM54P55 and KKKR44T45I46/RGDN54P55→SKAG44I46/RGDM54P55 in these domains, which are preceding the RGD loop and close flanking the RGD sequence. Thereby generating elegantin moledulars altered kistrin sequences.

1    Materials and Methods

1.1 Materials

A GST-kistrin clone was generously donated by Dr.Martin Humphries (University of Manchester, Manchester, U. K.) and the GST-elegantin clone was prepared as reported previously[15]. The primers were synthesized by GENSET SA in U. K.. All other chemicals and reagents were obtained from Sigma and Milligen (Watford, Herts, U. K.). They were at least analytic grade.

1.2 Site-directed mutagenesis and sequencing

Design and synthesize the site-directed mutation primers. For K41K42K43-R44T45I46/RGDM54P55→S41K42A43G44T45I46/RGDM54P55 mutagenesis, the primers are 5'-GCCGTTTCCTCAAATGCTGGACTATCTGCCGTCGTGCTCGTGGTGACCATCCGGACGACCG-3' (code) and 5'-CGGTCGTCCGGAGGTCACCACGAGCACGACGGCAGATAGTCCAGCATTTGAGGAAACGGC-3' (comp). For K41K42K43-R44I46/RGDM54P55→S41K42A43G44I46/RGDM54P55 mutagenesis, the primers are 5'-GCCGTTTCCTCAAATGCTGGATCTGCCGTCGTGCTCGTGGTGACCATCCGGACGACCG-3'(code) and 5'-CGGTCGTCCGGAGGTCACCACGAGCACGACGGCAGATCCAGCATTTGAGGAAACGGC-3'(comp). The PCR cycling parameters for site-directed mutation were going on following the instruction manual in QuikChangeTM Site-directed Mutagenesis Kit[16]. The base sequence assay of the mutated genes were going on according to the current protocols in molecular biology[17].

1.3 Gene expression and protein purification

The pGEX-3X vectors (Pharmacia) with the mutated elegantin genes (N107 or P107) were transformed into E. coli BL-21 according to the instruction manual in Epicurian Coli Competent and Supercompetent Cells[16]. The cells were cultured at 37 ℃ in LB liquid medium. When the cell density was to A＝0.8, IPTG(final concentration 0.1 mmol/L) was added in the culture. The cells were cultured continually for 2 h, then harvested the cells by centrifuge (2 500 g for 15 min). The supernatant was collected by 4 ℃ centrifuge (15 000 g for 30 min), after the cells were disrupted by sonicator. The GST-fusion proteins (Eleg-N107 and Eleg-P107) were batch purified using bulk Sepharose 4B[18].

1.4 Protein electrophoresis

SDS-PAGE, native PAGE and IEF were performed according to the Pharmacia method[19].

1.5 Platelet aggregation inhibition assay

Washed platelets were prepared from platelet rich plasma (PRP) obtained from healthy individuals who had not taken medication for nine days before bleeding. The GST-snake venom mutated proteins to washed platelets was performed as described previously[20]. Briefly, the incubation mixture was composed of 300 ml of washed platelets(3×108 per ml), 10 ml of agonist(1.75 mmol/L ADP) giving a final concentration of 50 mmol/L, 10 ml of GST-disintegrin protein and made to a final volume of 350 ml. The aggregation assay was allowed to procced for at least 120 s. The platelet aggregation was measured in Whole-blood AggregometerTM.

2 Results

2.1 DNA sequence and corresponding amino acid sequence of the mutated genes

Analyzing DNA sequences of the mutated kistrin and elegantin genes[21] and their corresponding amino acid sequences[21] were confirmed that the mutagenetion was perfomed according to our design(Fig.1).

 

Fig.1       Comparison of the base and amino acid sequence of wild-type and mutated disintegrins

 

2.2 The expressed and purified products of the mutated genes

E. coli (BL-21) including recombined pGEX-3X vectors was cultured in LB medium. As the cell grown to A＝0.8, IPTG was added to final concentration 0.1 mmol/L, The cells were cultured continually for 2 h, and then harvested the cells by centrifuge (2 500 g for 15 min). After the cells were disrupted by sonicator, the supernatant was collected by 4 ℃ centrifuge (15 000 g for 30 min). The GST-fusion proteins were batch purified using bulk Sepharose 4B. Assay shown that the mutated gene in recombinant pGEX-3X vectors could be well and soluble expressed in the competent cells BL-21 (Fig.2).

 

Fig.2       Expression and purification of the Eleg-N107 and Eleg-P107 genes products

1, marker proteins; 2-6, Eleg-N107; 7-11, Eleg-P107; 2,7, cell homology without induce by IPTG; 3,8, cell homology induced by IPTG for 2 h; 4,9, supernatant; 5,10, pellet; 6,11, elution by 25 mmol/L glutathione.

 

2.3 Eletrophoresis property analysis of the mutated proteins

On the SDS-PAGE, The four disintegrins mainly have two very similar bands in molecular weight. The lower bands are the degraded products of the original proteins [Fig.3(A)]. On the native-PAGE, the mutated proteins, Eleg-N107 and Eleg-P107 have weaker Rf 0.1-0.18 bands than the wild-type proteins, kistrin and elegantin. The mutated proteins Eleg-N107 and Eleg-P107 have several (Rf 0.67-1.00) bands, but the wild-type proteins kistrin and elegantin do not display. For the Rf 0.18-0.67, the wild-type proteins kistrin and elegantin have four bands respectively, but the mutated proteins Eleg-N107 and Eleg-P107 have only three bands [Fig.3(B)]. Their pI values are kistrin＞elegantin＞Eleg-P107＞Eleg-N107， respectively [Fig.3(C)].

 

Fig.3       Electrophoresis property compassion of the wild-type and mutated disintegrins

(A) SDS-PAGE; (B) native-PAGE; (C) IEF. 1, kistrin; 2, elegantin; 3, Eleg-N107; 4, Eleg-P107.

 

2.4 Inhibition of platelet aggregation by elegantin variants

It was found that the product of Eleg-N107 gene had dramatically reduced activity compared to elegatin as an inhibitor of platelet aggregation. In contrast, the product of Eleg-P107 gene strongly increased the activity relative to elegatin as an inhibitor of platelet aggregation (Fig.4).

 

Fig.4 Inhibition of the disintegrins Eleg-N107 and Eleg-P107 to ADP-induced platelet aggregation in PRP

 

3    Discussion

We have reported that the amino acid residues flanking the RGD motif in disintegrins have a major affect on their functional activities[15,22]. In the present report, we investigated the role of amino acids in the domains preceding the RGD loop and flanking RGD motif on biological function of the disintegrin elegantin. The results indicate that these changes KKKR44T45I46/A50RGDN54P55→SKAG44T45I46/P50RGDM54P55 and KKKR44T45I46/A50RGDN54P55→SKAG44I46/P50RGDM54P55 not only dramatically altered the activity of elegatin as an inhibitor of platelet aggregation, but also altered their electrophoresis properties. These observations suggest that these mutations alter the structure and function of the disintegrins, suggesting that these amino acid residues in the domains preceding the RGD loop and flanking RGD motif are important for structure and function of the disintegrins. Many studies found function of the disintegrins and GST-disintegrins were very similar in respect of inhibiting the ADP-induced aggregation[14,15]. Therefore, in this paper we used directly GST-disintegrins to study the importance of the amino acid residues flanking the RGD sequence for inhibiting the ADP-induced platelet aggregation.

Some studies showed that disintegrins and GST-disintegrins could bind each other in a solution and this bind could not be separated by native-PAGE[5,12], In present study, GST-kistrin, GST-elegantin, GST-Eleg-N107 and GST-Eleg-P107 of different molecular masses were found in the native-PAGE. It means that some properties of the elegantin variants are similar with wild-type elegantin and kistrin.

Comparison of biological activity of disintegrins Eleg-N107 and Eleg-P107 showed that the inhibitiory activity of Eleg-P107 for platelet aggregation dramatically increased 18-folds(Eleg-P107 IC50＝92 nmol/L; Eleg-N107 IC50＝1 650 nmol/L). Following the stucture models of kistrin and elegantin2a[5], the amino acid residues at 39-41 of kistrin and at 41-43 position of elegantin built an arm linking the consevative and variable area, and the amino acid residues at position 42-44 of kistrin and at 44-46 in elegantin formed a block positioned at the base of the RGD-loop, suggesting they play important roles in regulation of the disintegrin structure and function. When KKKR44T45I46/A50RGDN54P55 was changed into SKAG44T45I46/P50RGDM54P55, the variant structure was probably not similar with either elegantin or kistrin. e.g. the RGD loop structure was altered. Therefore, it was not able to bind on the aIibb3 complex with high affinity and to inhibit platelet aggregation. When KKKR44T45I46/A50RGDN54P55 was changed into SKAG44I46/P5RGDM54P55, structure of the RGD-loop was altered, which was more appropriate to binding with the aIibb3 complex than disintegrin Eleg-N107. Therefore, inhibitory activity of the Eleg-P107 in platelet aggregation assay was much higher than disintegrin Eleg-N107 and elegantin, but weaker than that of kistrin. These data suggests that the domain encompassing residues 38-47, located at the disintegrin core and preceding the RGD loop is important on disintegrin structrure-function.

 

Acknowledgements  We thank Martin Humphries for supplying the GST-kistrin vector.

 

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Received: September 8, 2000  Accepted: December 5, 2000

This work was supported by the National Natural Science Foundation of China, Grupo Grifols and Ceteon Respectory

*Corresponding author: Tel, 86-373-3326341; Fax, 86-373-3326524; e-mail, [email protected]

Abbreviations used: PRP, platelet rich plasma; Elegantin, platelet aggregation inhibitor from the venom of Trimerasurus elegans; Kistrin, inhibitor from the venom of Calloselasma rhodosto; EDTA, ethylene diaminetetra-acetic acid; IEF, isoelectric focusing.