In silico comparative protein analysis of human stem cell factor (SCF)

Dehbashi et al., J Appl Bioinform Comput Biol 2015, 4:2 http://dx.doi.org/10.4172/2329-9533.1000119

Journal of Applied Bioinformatics & Computational Biology

Research Article

In Silico comparative protein analysis of Human Stem Cell factor (SCF) Moein Dehbashi, Elahe Kamali and Sadeq Vallian*

well as several members of the ADAMs family including ADAM17 and ADAM33 have been reported to be responsible for cleavage of membrane-bound SCF [4-7]. In view of the reported significant roles for SCF in organisms development, we aimed to perform an in silico comparative analysis of the protein in human and the closest orthologous.

Materials and Methods SCF protein sequences

Abstract Stem cell factor (SCF) is a critical protein with several key roles in the cell such as hematopoiesis, gametogenesis and melanogenesis. In the present study, the in silico analysis including phylogenetics, protein network analysis (interactome) and conservation assessment on SCF was performed using NCBIBLAST, MEGA6 software, STRING 10 database and ClustalX 2.1 software. Analysis of the shared SCF protein interactors in human (Homo sapiens), Gorilla (Gorilla gorilla) and Chimpanzee (Pan troglodytes) demonstrated the presence of partly either conserved proteins interactors or proteins with the same function. Moreover, the data showed that 152 amino acids of SCF were conserved among the examined primates. Together, the results approved the gene flow and genetics similarity of SCF among human, Gorilla and Chimpanzee. This may suggest that during evolution, SCF gene transferred partly intact either on the basis of protein sequence or function from the same ancestors to Gorilla, Chimpanzee and human. Keywords: Stem cell factor; In silico protein analysis; Primates

Introduction Stem cell factor (SCF, also called Steel factor, Kit ligand or KITLG) is a growth factor that functions both as a membrane-bound and soluble forms [1]. Fibroblasts and endothelial cells express SCF protein, resulting in proliferation, migration, survival, and differentiation of hematopoietic progenitors, melanocytes, and germ cells [1]. The SCF gene is located on mouse (Mus musculus) chromosome 10, human (Homo sapiens) chromosome 12. It composed of ten exons in both human and mouse [2,3]. In human, the SCF protein undergoes two alternative splicing which result in soluble and membrane-bound isoforms (Figure 1) [1]. Both membrane-bound and soluble forms of SCF are regulated at the RNA and protein levels [1]. The isoforms vary only in exon 6, but encode membrane-bound SCF, containing an extracellular domain, a transmembrane domain, and an intracellular region [1]. The longer isoform is quickly cleaved to produce a 165-amino acid soluble SCF and the shorter isoform, lacking exon 6, forms membrane-bound type [1]. The receptor for membrane-bound and soluble SCF is the C-Kit protein with an intrinsic tyrosine kinase activity [1]. Proteases such as matrix metalloprotease-9, chymase-1 as *Corresponding author: Sadeq Vallian, Professor of Human Molecular Genetics, Genetics Division, Department of Biology, Faculty of Science, University of Isfahan, Isfahan, IR Iran, Tel: +98311793456; E-mail: svallian@sci. ui.ac.ir Received: August 08, 2015 Accepted: October 18, 2015 Published: October 25, 2015

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The sequences of human SCF (AC: NP_000890.1; isoform b precursor and AC: NP_003985.2; isoform a precursor) were extracted from NCBI database. The protein sequences were compared to all cellular life forms as well as human counterparts. Also, the intra- and interspecies sequences were compared in primates, and the phylogenic trees were constructed for all species.

Phylogenetics trees In this study bioinformatics tools including NCBI-BLAST tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and MEGA6 software [8] were used for sequence similarity search. The tools were also used for local alignments, i.e., the maximal regions of high similarity between the query sequence and the database sequences. The sequences were searched against all non-redundant protein sequences including GenBank, PDB, Swiss-Prot, protein information resource (PIR), protein research foundation (PRF) excluding environmental samples from whole genome shotgun (WGS) projects. The protein BLAST was used as the BLAST algorithm, as it compares a query to closely related sequences. In this way, very similar sequences were selected for alignment. In the next step, the BLAST results were used for

Figure 1: Schematic illustration of SCF splice forms and protein processing. A: SCF protein is synthesized as two transmembrane forms due to alternative splicing in exon 6, SCF220, and SCF248. In SCF248, exon 6 is remained and encodes a proteolytic cleavage site, generating the soluble SCF165. B: SCF220, lacking the cleavage site, forms membrane-bound SCF dimers (mSCF), and SCF248 is processed to SCF165 that forms soluble SCF (sSCF). Dashed lines display the SCF dimers that are held together by non-covalent interactions [18].

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Citation: Dehbashi M, Kamali E, Vallian S (2015) In Silico comparative protein analysis of Human Stem Cell factor (SCF). J Appl Bioinform Comput Biol 4:2

doi:http://dx.doi.org/10.4172/2329-9533.1000119 phylogenetic tree construction by means of definite methods. In addition, fast minimum Evolution and Neighbor Joining tools were used for the evaluation ray investigations of the data [9,10]. The Maximum sequence differences of 0.75 were used, and the maximum sequence differences larger than 0.5 were considered as accurate for sequence grouping as indicated by NCBI. Moreover, NCBI-based algorithms including Clustal W and muscle were used.

Protein network analysis (interactome) STRING 10 databases was employed (http://string-db.org) for protein network analysis (interactome) in comprehensive species database [11]. As protein network analysis, the human SCF isoforms and the results of NCBI-BLAST tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi) were considered to select and find closely evolved organisms having high protein conservation among all protein interactors with SCF. In this regard, active prediction methods including neighborhood, gene fusion, co-occurrence, co-expression, experiments, databases and text mining were chosen for direct (physical) and indirect (functional) associations of the SCF protein. Moreover, the medium confidence (0.4) and the rate of interactors (no more than 50 interactions) were selected for each isoform. The data were retrieved from Uniprot (http://www.uniprot.org/uniprot), and showed the involvement of SCF interactors in the biological processes obtained from reviewed (Swiss-Prot) and unreviewed data (TrEMBL) by gene ontology (http:// www.ebi.ac.uk/QuickGO) and reactome (http://www.reactome.org/).

Conservation assessment ClustalX 2.1 software was used to investigate the conserved and variable amino acid sequences of SCF among primates [12,13].

Results and Discussion It is assumed that the order of primates consists of the clade Euarchontoglires, which is placed within the clade Eutheria of mammalian class. From molecular evolutionary approach, Colugos (placental mammals) are more closely connected to primates [14]. Generally, primates have traditionally been divided into two suborders, Prosimii and Anthropoidea [15]. This is a gradistic classification, because Prosimians are a grade. They are identified by their retention of primitive anatomical features [15]. In contrast to Prosimians, Anthropoids are also a clade, or natural phyletic unit. Because the features that distinguish Anthropoids from other primates are unique to Anthropoids, and are derived with respect to other primates [15]. There are three major radiations of extant Anthropoids, or higher primates: i) the Platyrrhines, or New World monkeys, from south and central America; ii) two groups of Catarrhines from Africa, Europe, and Asia—the Cercopithecoids, or Old World monkeys; iii) the Hominoids, or apes and humans [15]. The Platyrrhine monkeys are the only primates in the Neotropics, and they fill a diverse array of ecological niches there [15]. In the Old World, there are two very distinct radiations of higher primates that make up the infraorder Catarrhini—the Old World monkeys (Cercopithecoidea) and the Hominoids (Hominoidea) [15]. The Hominoids are restricted to a few species from the tropical forests of Africa and Asia and one cosmopolitan species—humans [15]. Our BLAST results of the isoform b precursor of human SCF (AC: NP_000890.1) by NCBI-BLAST tool (http://blast.ncbi.nlm.nih.gov/ Blast.cgi) showed that human SCF was placed in primates cluster, and was close to placental but far from rodents (Figure 2). Among primates, BLAST analysis of the protein sequence of human SCF

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Figure 2: Phylogenic analysis of SCF protein sequences. BAST analysis and phylogenetic tree of protein sequences of SCF using NCBI BLAST tree view, Neighbor joining clustering method and Max Seq Difference 0.75.

by MEGA6 software [8] showed that both human SCF isoforms were placed in the cluster of human and apes. It was demonstrated that human SCF isoform b was placed in the cluster along with Pan paniscus. Moreover, these results showed that human SCF isoform was placed in the cluster along with three SCF isoforms of Gorilla gorilla (Figure 3). MEGA6 analysis demonstrated that human and apes main cluster was close to that of old world monkeys. However, SCF protein of prosimians was far from those of human and apes, old world monkeys and new world monkeys. This classification approved previous systematic studies [16,17] (Figure 4). Based on the data of the SCF isoforms from the phylogenetic tree, it was indicated that three primates including Aotus nancymaae, Callithrix jacchus and Gorilla gorilla had three isoforms, but old world monkeys and prosimians had two SCF isoforms. However, only one prosimian called Otolemur garnettii had five SCF isoforms. This previous primate classification showed that humans (Homo genus) and Chimpanzees (Pan genus) originated from a common ancestor that was far from other Hominidae such as Gorilla and Orangutan (Figure 4). Moreover, it could be expected that Hominidae family would be composed of Homininae (Homo, Pan and Gorilla genera) and Ponginae subfamilies, including Orangutan (Pongo abelii). These results were consistent with the accepted evolutionary relationships of the Hominoidea [18]. Traditionally, apes have been divided into the lesser apes (Hylobatidae family) and the great apes (Pan, Gorilla and Pongo genera) [19,20]. The classification of Strepsirrhini and Haplorhini suborders of primates was introduced by Anderson et al [21], and subsequently pursued by McKenna et al [17] (Figure 4) [17,22,23]. According to this hypothesis, the primates were divided into two superfamily: Prosimii and Anthropoidea [24]. Prosimii included all Prosimians: Strepsirrhini plus Tarsiers and Anthropoidea contained all simians [24]. In addition, modern monophyletic classifications used groups that were monophyletic [25]. A molecular phylogeny analysis by Springer and colleagues (2012) for 70 primate genera and 367 primate species was conducted based on a concatenation of 69 nuclear gene segments and ten mitochondrial gene sequences, most of which were extracted from GenBank [1]. It was suggested that living primates • Page 2 of 6 •

Citation: Dehbashi M, Kamali E, Vallian S (2015) In Silico comparative protein analysis of Human Stem Cell factor (SCF). J Appl Bioinform Comput Biol 4:2

doi:http://dx.doi.org/10.4172/2329-9533.1000119 genes encoding proteins with DUF1220 domains in humans (Rogers and Gibbs 2014). This might be related to the expansion of human brain size [26].

Figure 3: BLAST analysis of the protein sequence of human SCF using MEGA6 software.

Figure 4: Phylogenetic tree of primates proposed by McKenna and Bell in 1997 (McKenna and Bell 1997).

shared a common ancestor 71–63 Ma, and that divergences within both Strepsirrhini and Haplorhini were entirely post-Cretaceous [14]. In the present study, our results were found to be consistent with those obtained by Springer and colleagues (2012) in placing human close to Pan based on the genetic relationship in the clade of Hominidae [25]. In addition, cDNA BLAST analysis showed that some primates possessed copy number change and duplication [26]. The majority of protein-coding genes have 1:1 homologues among humans, the great apes and old world monkeys sequenced so far, but gene content was not identical among primate species [26]. Particular gene families have been expanded or contracted in individual lineages (Rogers and Gibbs 2014). For example, 1,358 genes were identified as new duplications in the Rhesus macaque genome and compared with the human genome [26]. For instance, the major histocompatibility complex (MHC) gene cluster, which is critical for response to pathogens and other immunological processes, has been expanded in macaques, relative to humans [26]. Interestingly, changes in genes encoding zinc-finger transcription factors, which show gains and losses, had greatly distinguished the genomes of humans, Chimpanzees and Orangutans, as well as the marked expansion of Volume 4 • Issue 2 • 1000119

Nevertheless, the draft quality of current non-human primate genome assemblies makes it difficult to define all copy-number variations accurately [26]. The present data suggested that human and Chimpanzees underwent more rapid changes in gene copy number than Orangutans and Rhesus macaques [26]. Among the great apes, Gorillas showed more copy-number variants than others [26]. However, complete analyses await additional data, including better genome assemblies and information concerning copynumber polymorphism in non-human primates [26]. Segmental duplications (that is, chromosomal regions >1 kb, that are >90% identical to other segments in the same genome) are a significant aspect of primate genome structure and dynamics. Duplication and deletion of these segments are active in the human genome [26]. Some of these mutations are apparently neutral, but many may lead to adverse consequences and diseases. Similar to the way in which segmental duplications create variation among humans, they could function as ‘drivers’ of evolutionary change across primate genomes [26]. About 5% of the human and Chimpanzee genomes, and 3.8% of the Orangutan genome, comprise segmental duplications [26]. The genomes of human and great ape are enriched with dispersed duplications, as they have been subjected to an interval after their divergence from old world monkeys, when the production of new duplications was particularly active [27,28]. Based on our BLAST analysis, we showed that SCF during evolution had no duplication, but the variable number of isoforms among some primate might be obtained from alternative splicing variations. It is interesting to note that similar results were obtained using NCBI-based algorithm, clustalW, and muscle algorithm of MEGA6 software (data not shown) [22]. Protein network analysis (interactome) using STRING 10 database further demonstrated protein intractors of human SCF (Figure 5). Our hypothesis for performing this analysis was that the NCBI-BLAST tool could find and demonstrate only the orthologous sequences with high similarity in comparison with the query sequence. However, a protein in an organism can involve in several pathways and interact with several protein interactors. From the evolutionary view, when the developmental roles of a protein are investigated among all available cellular life forms, it is necessary to demonstrate the same functions by protein interactors and find closely evolved organisms. In the present study, according to the results of protein similarity using MEGA6, Chimpanzee and Gorilla SCF in STRING 10 database was selected to compare the protein interactors with human. According to these data, many protein interactors of H. sapiens, P. troglodytes and Gorilla gorilla SCF showed conservation (Figure 6). To fulfill our hypothesis, shared and unique protein interactors between human, P. troglodytes and Gorilla gorilla SCF by the comparison of SCF interactors were obtained (Table 1). Moreover, SCF shared and unique protein interactors of H. sapiens, P.troglodytes and Gorilla gorilla from Uniprot (http://www.uniprot.org/uniprot) were retrieved. The data showed the involvement of them in the biological processes for reviewed data (Swiss-Prot), the unreviewed data (TrEMBL) by gene ontology (http://www.ebi.ac.uk/QuickGO) as well as reactome (http://www.reactome.org). All gene ontologies and pathways resulting from H. sapiens, P. troglodytes and Gorilla gorilla showed that these proteins played shared significant roles, for example,

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Citation: Dehbashi M, Kamali E, Vallian S (2015) In Silico comparative protein analysis of Human Stem Cell factor (SCF). J Appl Bioinform Comput Biol 4:2

doi:http://dx.doi.org/10.4172/2329-9533.1000119

Figure 5: Protein network analysis (interactome) of human SCF using STRING 9.1 database. \

Figure 6: Occurrence view of protein interactors to identify the conservation of protein interactors between human, Chimpanzee and Gorilla SCF.

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Citation: Dehbashi M, Kamali E, Vallian S (2015) In Silico comparative protein analysis of Human Stem Cell factor (SCF). J Appl Bioinform Comput Biol 4:2

doi:http://dx.doi.org/10.4172/2329-9533.1000119 Table 1: Comparison of protein interactors of SCF between H. sapiens, G. gorilla and P. troglodytes using STRING 10. Unique protein interactors of H. sapiens SCF

Unique protein interactors of G. gorilla SCF

Unique protein interactors of P. troglodytes SCF

Shared protein interactors among H. sapiens, P. troglodytesn and G. gorilla SCF

MAPK8

INSR

CMA1

SNAI2

AKT1

EPOR

SOCS1

GATA1

GDF9

KIT

DOK1

JAK2

GRB10

GRB2

SOS1

MATK

CDC6

MYB

MCM2

SHC1

ORC5

STAT3

ORC1

PIK3R1

MCM7

HRAS

MCM6

LYN

CDT1

FLT3LG

PTPRO

AMH

STAP1

TEC

ORC4

PIK3CA

GAB1

FGF7

MCM4

KDR

ORC3

CSF1R

ORC2

cytokine-mediated signaling pathway, erythropoietin-mediated signaling pathway, female gamete generation, regulation of MAPK cascade, axon regeneration, erythrocyte differentiation, leukocyte migration, axon guidance, cell proliferation, cell differentiation, cell migration, positive regulation of T-helper cell differentiation, B cell differentiation, nervous system development, T cell co-stimulation, genitalia development and etc. Moreover, unique SCF protein interactors of Gorilla gorilla icluding INSR, AKT1 and GATA1 showed their involvement in the biological processes such as activation of MAPK activity, cellular response to growth factor stimulus, positive regulation of developmental growth, germ cell development, male sex determination, regulation of embryonic development, embryonic hemopoiesis, male gonad development, erythrocyte development and etc. There is only one unique SCF protein interactor of Pan troglodytes named CMA1. This protein involved in proteolysis, protein processing, interleukin-1 beta biosynthetic process, serine-type endopeptidase activity, extracellular space and intracellular. Also, unique protein interactors of human SCF involved in, for example, apoptotic signaling pathway, pigmentation, cytokine-mediated signaling pathway, cell surface receptor signaling pathway, negative egulation of phosphorylation, leukocyte migration, cell division, DNA replication, cellular response to epidermal growth factor stimulus, axon guidance, G1/S transition of mitotic cell cycle, epidermal growth factor receptor signaling pathway, fibroblast growth factor receptor signaling pathway and etc. Theses retrieved data and the number of shared protein interactors partly showed a large number of conserved biological processes pertained to SCF across H. sapiense, P. troglodytes and Gorilla gorill. Furthermore, by using ClustalX 2.1 software [29], we demonstrated the conserved and variable amino acid sequences of SCF among primates. The results showed that 152 amino acids of SCF were remained conserved across considered primates (Figure 7). Generally, by utilizing these methods at the protein level, we obtained novel data regarding SCF key gene and explained the genetic relation between modern human and primates. All results approved the gene flow and genetics similarity of SCF among human, P. troglodytes and Gorilla gorilla [30]. In short, it seems that during the Volume 4 • Issue 2 • 1000119

Figure 7: Conserved and variable amino acids sequences of SCF across the considered primates using ClustalX 2.1 software [12, 13]. Part of SCF sequence has been illustrated.

evolution, SCF gene as a key gene, was transferred partly intact either on the basis of sequence or function from the same ancestors to P. troglodytes, Gorilla gorilla and modern human. Acknowledgments This study was supported by department of research of University of Isfahan, Isfahan, IR Iran. The authors declare that there is no conflicts of interest.

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Citation: Dehbashi M, Kamali E, Vallian S (2015) In Silico comparative protein analysis of Human Stem Cell factor (SCF). J Appl Bioinform Comput Biol 4:2

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Author Affiliation

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Division of Genetics, Department of Biology, Faculty of Science, University of Isfahan, Isfahan, IR Iran

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