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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Result and discussion
  6. Conclusion
  7. Acknowledgements
  8. References

Granulocyte–macrophage colony-stimulating factor (GM-CSF) is a cytokine that is essential for growth and development of progenitors of granulocytes and monocytes/macrophages. In this study, we report molecular cloning, sequencing and characterization of GM-CSF from Indian water buffalo, Bubalus bubalis. In addition, we performed sequence and structural analysis for buffalo GM-CSF. Buffalo GM-CSF has been compared with 17 mammalian GM-CSFs using multiple sequence alignment and phylogenetic tree. Three-dimensional model for buffalo GM-CSF and human receptor complex was built using homology modelling to study cross-reactivity between two species. Detailed analysis was performed to study GM-CSF interface and various interactions at the interface.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Result and discussion
  6. Conclusion
  7. Acknowledgements
  8. References

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a glycoprotein that is required for the production of granulocytes and macrophages from bone marrow. It is also a potent hematopoietic growth factor that stimulates proliferation and maturation of myeloid progenitor cells. GM-CSF has been shown to increase the immune response both in animal model and in clinical trials, irrespective of its antigenic source (DNA, protein, peptide and antigen loaded dendritic cells) (Disis et al., 1996; Slingluff et al., 2003). It can act as a potent adjuvant for the immunization. It has been used as an effective adjuvant with VP1 recombinant protein for foot and mouth disease (Zhang et al., 2011).

The GM-CSF receptor is composed of alpha and beta subunits. The α-subunit binds GM-CSF with low affinity (Kd = 1–5 nm), and β-subunit forms heterodimer complex with α-subunit, which results in a high-affinity (Kd = 40–100 pm) receptor that is capable of transmitting growth signals (Chiba et al., 1990; Hayashida et al., 1990; Kitamura et al., 1991; Park et al., 1992). The α- and β-receptors are proved to be required for the protective immunization in a cell-based antitumor vaccination (Zarei et al., 2009). GM-CSF receptor activation follows general rules that invoke receptor dimerization and tyrosine transphosphorylation of the cytoplasmic domains (Schlessinger, 2000). The recent structural study showed that the GM-CSF/GM-CSF receptor forms ternary complex and it assembles an unexpected dodecamer arrangement (Hansen et al., 2008).

Cloning and characterization of bovine, murine, ovine and human GM-CSF cDNA have already been reported (Gough et al., 1984; Wong et al., 1985; Leong et al., 1989; O'Brien et al., 1991). Although the identities between these genes are relatively high, the biological activities of these proteins are generally species specific in their actions, the exception being human GM-CSF (hGM-CSF), which has limited activity on bovine bone marrow cells (Cantrell et al., 1985; Leong et al., 1989). This species-specific restriction will require the availability of purified or recombinant GM-CSF for each economically important species to examine both therapeutic and adjuvant potentials of this factor. Buffalo is one of the most economically important animal for its contribution to milk production in tropical countries; however, the buffalo GM-CSF cDNA has not yet been cloned. In this study, we report the primary coding sequence of the buffalo GM-CSF (bGM-CSF) and compare it with those of humans, bovines and other animals. A bioinformatics approach combining sequence analysis, secondary structure prediction, homology modelling and interface analysis has been used to study bGM-CSF and its interactions with human GM-CSF receptors.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Result and discussion
  6. Conclusion
  7. Acknowledgements
  8. References

Isolation and primary culture of PBMCs

Blood samples of adult apparently normal Indian water buffaloes were collected from the University Livestock farm. Peripheral blood mononuclear cells (PBMCs) were prepared by Histopaque 1.088 (Sigma, St. Louis, MO, USA) density gradient centrifugation of heparinized blood. The cells were washed thrice in phosphate-buffered saline (pH 7.2) and resuspended in RPMI-1640 medium containing 10% foetal bovine serum (Gibco BRL, Life Technologies, Bethesda, MD), 20 mm HEPES and 2 mm L-glutamine at the concentration of 5 × 106 viable cells mL−1. Concanavalin A was added at a concentration of 10 μg mL−1 to stimulate PBMCs and incubated at 37°C for 24 h with 5% CO2 in a humidified incubator.

RNA isolation and cDNA cloning

The viable cells enumerated by trypan blue exclusion and total cellular RNA were isolated from 1 × 108 cells as per the standard procedure (Sambrook et al., 1989). cDNA was synthesized using Moloney murine leukaemia virus reverse transcriptase and an oligo dT primer. Five microlitres of the above-synthesized cDNA was used for the GM-CSF amplification. The primers for the coding region of the buffalo GM-CSF were designed from the cattle GM-CSF sequence (GenBank accession no. NM_17402). The sequences of the primers were as follows: GM-CSFF: 5′ ATGTGGCTGCAGAAC 3′ and GM-CSFR: 5′ TCACTTCTGGGCTGG 3′. The amplified PCR product was cloned in pTZ57R/T vector (MBI Fermentas, Burlington, Canada), and positive clones were sequenced by Sanger sequencing method.

Sequence analysis

Amino acid sequence (bGM-CSF) was deduced from the nucleotide sequence using Lasergene software (DNASTAR, Madison, WI). The physicochemical properties of bGM-CSF were computed using ProtParam tool, which is a part of ExPasy server (http://us.expasy.org/tools/protparam.html) (Gasteiger et al., 2005). Signal peptide was predicted using SignalP, version 3.0 (Jannick et al., 2004). N-glycosylated sites are predicted using NetNGlyc server (http://www.cbs.dtu.dk/services/NetNGlyc/).

Multiple sequence alignment and phylogenetic analysis

To perform multiple sequence alignment, we obtained homologous sequences for bGM-CSF using BLAST search against Swiss-Prot database with an E value of 0.001 (Altschul et al., 1997). CLUSTALW was employed to generate multiple sequence alignment of bGM-CSF and other GM-CSF proteins (Chenna et al., 2003). Phylogenetic tree was created using phylip package (Felsenstein, 1989).

Model building and refinement

Three-dimensional model for bGM-CSF was built using homology modelling software, MODELLER (Eswar et al., 2006). Human GM-CSF (PDB code: 3CXE, chain:B) was selected as a structural template. The pairwise alignment between bGM-CSF and hGM-CSF was created using CLUSTALW (Thompson et al., 1994). Of 20 homology models generated, the model with lowest energy was selected as a final model for further analysis. To assess the quality of the model, we validated the final model using HARMONY server, which assesses the compatibility of an amino acid sequence with a proposed three-dimensional structure (Pugalenthi et al., 2006).

Buffalo GM-CSF–human receptor complex

We employed two steps to model bGM-CSF and receptor complex. In the first step, we used LSQMAN program to superimpose bGM-CSF on top of hGM-CSF, which is a part of receptor complex (PDP code 3CXE) (Kleywegt & Jones, 1994). In the second step, human GM-CSF was removed from the superimposed coordinates. This gave us bGM-CSF–receptor complex (bGMR complex) containing buffalo GM-CSF, human alpha receptor and human beta receptor.

Interface analysis

The interface between bGM-CSF and human receptors was analysed using protein interactions calculator (PIC) server which, given the coordinate set of 3D structure of a protein or an assembly, computes various interactions such as disulphide bonds, interactions between hydrophobic residues, ionic interactions, hydrogen bonds, aromatic–aromatic interactions, aromatic–sulphur interactions and cation–π interactions within a protein or between proteins in a complex (Tina et al., 2007). Interface residues and accessible surface area (ASA) of interface residues were calculated using InterProSurf server (Negi et al., 2007).

Result and discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Result and discussion
  6. Conclusion
  7. Acknowledgements
  8. References

Buffalo GM-CSF sequence

The nucleotide sequence of buffalo GM-CSF cDNA containing a 432-bp open reading frame (ORF) coding for 143 amino acids has been submitted to GenBank (accession number AY553190) (Fig. 1). The deduced amino acid sequence of bGM-CSF has an estimated mass of 16 105 Da. The bGM-CSF contains signal peptide at N-terminal end (position 1-17). Two glycosylation sites occur at positions 44 and 54. Matured bGM-CSF contains four cysteine residues at positions 70, 104, 112 and 137. Scanning of PROSITE database shows that this sequence contains granulocyte–macrophage colony-stimulating factor signature C-P-[LP]-T-x-E-[ST]-x-C (de Castro et al., 2006).

image

Figure 1. The cDNA nucleic acid sequence encoding the buffalo GM-CSF (GenBank Database accession number AY553190). The forward and reverse primer regions are underlined. The corresponding GM-CSF deduced amino acid sequence (bottom line) is also shown. The putative signal peptide (1–17 amino acids) is bolded and marked (Δ).

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The physicochemical properties of bGM-CSF were computed using ProtParam tool (Gasteiger et al., 2005). bGM-CSF contains 12 positively charged amino acids, 16 negatively charged amino acids, 45 hydrophobic amino acids and 44 polar amino acids. The calculated isoelectric point for bGM-CSF is 5.309. This indicates that bGM-CSF is acidic in nature. The GC content is 56.02%. ProtParam assigns instability index for protein to classify whether the protein is stable or unstable. A protein whose instability index is smaller than 40 is predicted as stable; a value above 40 predicts that the protein may be unstable (Guruprasad et al., 1990). The computed instability index for bGM-CSF is 45.78. This result shows bGM-CSF is a slightly unstable protein. The aliphatic index (AI), which is defined as the relative volume of a protein occupied by aliphatic side chains (A, V, I and L) is regarded as a positive factor for the increase in thermal stability of globular proteins. Aliphatic index for bGM-CSF is 74.34. The very high aliphatic index indicates that bGM-CSF may be stable for a wide temperature range.

Sequence comparison

Buffalo GM-CSF was aligned with other GM-CSFs from 17 different animals using CLUSTALW (Fig. 2). As seen in Fig. 2, the comparison of the protein sequence of GM-CSF with those of the other GM-CSF reveals that they are closely related to each other. Human and buffalo GM-CSFs display 70% similarity. Of 143 amino acids, 103 are identical. Four cysteines, which are conserved in all the members (alignment positions 71, 105, 113 and 138), form two conserved disulphide bonds (alignment positions 71-113 and 105-138). N-glycosylated asparagine at positions 44 and 54 is conserved in all the animals except in Otolemur garnettii and Ovis aries where asparagine is replaced by aspartate. Glycosylation is essential for GM-CSF activity. It has been reported that fully glycosylated GM-CSF is biologically more active in vivo than the nonglycosylated protein (Forno et al., 2004). The GM-CSF protein is 144 amino acids long in most of the animals. But GM-CSFs in Bubalus bubalis, Bos taurus and Otolemur garnettii is 143 amino acids. A gap occurs at 56th position in Otolemur garnettii and 57th position in Bubalus bubalis and Bos taurus. The longest one is Equus caballus that contains 146 amino acids. All GM-CSF sequences contain conserved granulocyte–macrophage colony-stimulating factor signature C-P-[LP]-T-x-E-[ST]-x-C (Fig. 2).

image

Figure 2. Multiple sequence alignment of buffalo GMCSF with other GMCSFs. Granulocyte-macrophage colonystimulating factor signature is marked with rectangular box. Interface residues are shown in bold letters. Positions with identical, very similar, similar amino acids are marked with “*”, “:” and “.” respectively. H and E represent alpha helices and beta strand respectively.

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A phylogenetic tree has been constructed using multiple sequence alignment of 17 GM-CSF sequences from different animal species; result shows that GM-CSFs from both buffalo (Bubalus bubalis) and human is derived from common ancestors, but they are far from each other according to evolutionary point of view; it shows that they belong to different orders (Fig. 3).

image

Figure 3. Phylogenetic analysis of buffalo GM-CSF and GM-CSF from 17 mammalian species.

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Homology model of buffalo GM-CSF

The homology model of bGM-CSF was obtained using 3.3 A° resolution structure of human GM-CSF–receptor complex (hGMR complex) (Hansen et al., 2008) (PDB ID: 3CXE, chain: B) (Helen et al., 2000). MODELLER was employed to build homology model. The quality assessment of the model using HARMONY server shows that the model has good quality with no local or global error. DSSP was employed to identify the secondary structural features of bGM-CSF and hGM-CSF (Kabsch & Sander, 1983). hGM-CSF has 5 α-helices and 2 β-sheets with two residues length, whereas bGM-CSF has five α-helices and one β-sheet. The absence of one beta sheet in bGM-CSF is due to the deletion of one amino acid, which is a part of first beta strand (Fig. 2).

Analysis of GM-CSF–receptor complex

So far, no receptor sequences for buffalo GM-CSF have been reported. Therefore, we used human GM-CSF receptors (PDB code: 3CXE; chain:A and chain:C) to build bGM-CSF and human receptor (bGMR) complex (Fig. 4). The bGMR and hGMR complexes were analysed using InterProSurf server and PIC server (Tina et al., 2007). The result is shown in Tables 1 and 2. Sixteen interface residues (VAL 16, ASN 17, ILE 19, GLN 20, GLU21, ARG23, LEU25, LEU 49, LYS72, GLY75, PRO 76, MET 79, LEU 114, LEU 115, ILE 117 and PRO 118) were identified in hGM-CSF, whereas only 10 interface residues (VAL 33, ALA 35, LEU42, SER88, GLY 91, SER92, MET 95, PHE 131, ILE 133 and PRO 134) were identified in bGM-CSF. Accessible surface analysis shows that most of the residues in contact have accessible surface area in the range of 0–20%. This indicates that the interacting residues are buried, whereas a fraction of residues are exposed. The important interactions identified as crucial for maintaining of the three-dimensional structure of a GMR complex are hydrogen bonds and hydrophobic forces. As seen in Table 1, hGMR complex has more number of hydrophobic interaction and hydrogen bonds than bGMR complex. This indicates that hGMR complex is more stable than bGMR complex.

Table 1. Interactions between buffalo GM-CSF and human receptor
Molecule 1PositionResidueMolecule 2PositionResidueInteraction type
bGM-CSF33VALAlpha248TYRHydrophobic
bGM-CSF131PHEAlpha303ILEHydrophobic
bGM-CSF134PROAlpha246LEUHydrophobic
bGM-CSF42LEUBeta104VALHydrophobic
bGM-CSF35ALABeta105VALHydrophobic
bGM-CSF95METBeta105VALHydrophobic
bGM-CSF38GLUBeta105VALHydrogen bond
bGM-CSF37LYSAlpha301VALHydrogen bond
bGM-CSF130LEUAlpha302ARGHydrogen bond
bGM-CSF131PHEAlpha302ARGHydrogen bond
bGM-CSF133ILEAlpha302ARGHydrogen bond
bGM-CSF134PROAlpha302ARGHydrogen bond
bGM-CSF38GLUBeta39TYRHydrogen bond
bGM-CSF37LYSAlpha250ASPSalt bridge
Table 2. Interactions between human GM-CSF and human receptor
Molecule 1PositionResidueMolecule 2PositionResidueInteraction type
hGM-CSF16VALAlpha248TYRHydrophobic
hGM-CSF115LEUAlpha303ILEHydrophobic
hGM-CSF118PROAlpha246LEUHydrophobic
hGM-CSF18ALABeta105VALHydrophobic
hGM-CSF25LEUBeta104VALHydrophobic
hGM-CSF76PROBeta104VALHydrophobic
hGM-CSF76PROBeta105VALHydrophobic
hGM-CSF79METBeta105VALHydrophobic
hGM-CSF23ARGAlpha302ARGHydrogen bond
hGM-CSF114LEUAlpha302ARGHydrogen bond
hGM-CSF115LEUAlpha302ARGHydrogen bond
hGM-CSF117ILEAlpha302ARGHydrogen bond
hGM-CSF21GLUBeta105VALHydrogen bond
hGM-CSF72LYSBeta102SERHydrogen bond
hGM-CSF72LYSBeta107ASPSalt bridge
image

Figure 4. Three dimensional model of bGM-CSF and human receptor complex. α and β represents α and β receptors.

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Alpha receptor has four crucial interface residues (Leu 246, Tyr 248, Arg 302 and Ile 303) that interact with GM-CSF. Our analysis shows that both GM-CSFs establish similar interactions with those four crucial residues (Tables 1 and 2). The crucial residues in beta receptor that play role in GM-CSF interaction are Ser 102, Val 104 and Val 105. In hGMR complex, Val 104 forms hydrophobic interaction with Lue 25 (helix 1), pro 76 (helix 4) of hGM-CSF and in bGMR complex, Val 104 interacts with Leu 42 (helix 1) of bGM-CSF. It also makes hydrogen bond with Glu 38 in helix 1 of bGM-CSF. Val 105 forms hydrophobic interactions with Pro 76 (helix 1), Met 79 (helix 4) in hGM-CSF and Ala 35 (helix 1) and Met 95 (helix 4) of bGM-CSF. In addition, Val 105 form hydrogen bonds with Glu residues (helix 1) of both GM-CSFs. Lys 72 (helix 4) of hGM-CSF forms a salt bridge with Asp 107 of beta receptor. This salt bridge is absent in bGM-CSF where Lys is replaced by Ser. A salt bridge is observed between Lys 37 (helix 1) of bGM-CSF and Asp 250 of alpha receptor. This salt bridge is missing in hGM-CSF due to the absence of Lys, which is replaced by Gln.

From our analysis, it is observed that helix 4 of GM-CSF plays a role in beta receptor interaction, whereas helix 5 plays a role in alpha receptor interaction. Helix 1 of GM-CSF interacts with both alpha and beta receptors. The previous study showed that bovine GM-CSF has weak activity in both humans and mice (Charles et al., 1988). As bovine and buffalo GM-CSFs share 92% sequence identity, we can also expect a weak bGM-CSF activity in humans. It is obvious from our analysis that most of the interface residues that play a role in receptor binding are conserved in both human and buffalo GM-CSFs. Very similar interactions were observed in both hGMR and bGMR complexes. This indicates that bGM-CSF might interact with human GM-CSF receptors.

Conclusion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Result and discussion
  6. Conclusion
  7. Acknowledgements
  8. References

Buffalo cytokines have potential application in livestock industries. Buffaloes are more tolerant to tropical infection than bovines. But buffalo calves are more susceptible to FMD, pasteurellosis, tuberculosis, brucellosis and buffalo pox infections. GM-CSF, an important hematopoietic growth factor and immune modulator, directly involves in the control of innate resistance and therefore has the potential to boost immune responsiveness in immunosuppressed animals. FMD vaccination with bovine GM-CSF has shown encouraging results in bovine [3]. Similarly, buffalo GM-CSF can act as a good adjuvant for vaccination in buffaloes. The design of successful vaccination may also therefore benefit from the incorporation of GM-CSF. The present study describes cloning, sequence analysis, prediction of three-dimensional structure and structural analysis of the deduced amino acid sequence of the Indian water buffalo GM-CSF. Sequence and structural analysis showed that bGM-CSF is closely related to hGM-CSF. Both human GM-CSF and buffalo GM-CSF have similar interface residues that are conserved in both humans and buffaloes. Our study shows that residues in helix 1, helix 4 and helix 5 play a role in receptor interaction. Very similar interactions observed in both bGMR and hGMR complexes suggest that bGM-CSF can interact with human GM-CSF receptors.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Result and discussion
  6. Conclusion
  7. Acknowledgements
  8. References

The authors thank the Department of Biotechnology (DBT), Government of India, for the financial assistance to this Project. ST and MHS were SRFs who worked in this project. GP carried out the bioinformatics analysis.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Result and discussion
  6. Conclusion
  7. Acknowledgements
  8. References
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