VARIABILITY OF MESENCHYMAL VEGFR 1, 2, AND 3 PROTEIN EXPRESSION OFHUMANS AND MURINE SPECIES-A COMPARATIVE ANALYSIS

Ahmed Alrabai 1 , Ahmed AL-Hakami 1,2 and Harish C Chandramoorthy 1, 2 . 1. Center for Stem Cell Research, College of Medicine, King Khalid University, Abha, Saudi Arabia 2. Department of Microbiology and Parasitology, College of Medicine, King Khalid University, Abha, Saudi Arabia ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

The Vascular Endothelial Growth Factor 1,2,3 (VEGFR 1,2,3) protein sequences from various species like Mus musculus (Mouse), Rattus norvegicus (Rat) and Homo sapiens (Human) were retrieved from SWISS PROT database and compared for their homology and variability.DNAMAN Software was used to create multiple sequence alignment and in assessment of the homology and variability.The results of the phylogenetic tree assessment for VEGFR1, 2, 3 depicts that the sequences from Mus musculus (Mouse) and Rattus norvegicus (Rat) showed high homology when compared with the sequences of the Homo sapiens (Human). The intra sequence homology within the humanVEGFR1,2,3 protein predicted and analyzed for secondary structure show a characteristic variability in the amino acid positions on the random coils, alpha confirmationsand extended strands without significant variation in the length.

…………………………………………………………………………………………………….... Introduction:-
Stem cells are totipotent primordial cells capable to differentiate into any known adult cell types with its typical physiological functions (Louveau I et al., 2016).The first stem cells originate within the developing embryo (blastocyst) and have two important characteristics that distinguish them from other types of cells. First, they are unspecialized cells that renew themselves for long periods through cell division. The second is that, under physiologic or experimental conditions, they can be induced to become cells with special functions such as the beating cells of the heart muscle or the insulin-producing cells of the pancreas (Adel Alhadlaq and Jeremy J. Mao, 2004).Stem Cells reside as resident population at various organs and can be isolated from variety of tissues like adipose tissue, bone marrow, embryos, olfactory ensheathing cells, peripheral blood, skin and umbilical cord blood.Further stem cells or progenitor cells can be mobilized form bone marrow upon signaling from the infracted or damages tissues.
There are different types of stem cells like embryonic stem cells, endothelial stem cells, mesenchymal or stromal stem cells, hematopoietic progenitors, cardiac, cancer and neuronal stem cells, induced pluripotent stem cells etc (Choudhery MS et al., 2015). All these have been isolated, banked or used for research with few successful clinical applications. However identification of these cell with their characteristic phenotypic or genotypic markers are more difficult and challenging today. There are surface markers common for these cells with few 1324 exceptions like common leucocyte markers like CD45 which is mostly observed only in the cells of the hematopoietic origin (Kobolak et al., 2016). Further many progenitor stem cells usually shares surface markers making it difficult to isolate or characterize as functional lineages, though these markers are very particular for some discrete functional adult differentiated cells. Therefore the identification and characterization of the stem cells with the available markers or cluster of differentiation (CD) on the surface of these naïve cells becomes more difficult and generalized rather than specific. Therefore it becomes necessary to eliminate such older cells based on the other markers characteristic to the anatomical site of the cells or type of the cells.The best example of these cases isVascular Endothelial Growth Factor(VEGFR) group of the markers which are very specific to endothelial progenitors or endothelial cells (Igarashi Y et al., 2016). The presence of VEGFR has been well characterized in the mesenchymal stem cells too (Totsugawa T et al., 2002), though MSCs functions are much varied.The VEGFRfamily consists of three members, Vascular Endothelial Growth Factor Receptor -1 (Flt-1), Vascular Endothelial Growth Factor Receptor -2 (KDR/Flk-1) and Vascular Endothelial Growth Factor Receptor -3 (Flt-4), all of which belong to the receptor type tyrosine kinase superfamily (Matthias Clauss, et al., 1996, Kasper G et al., 2007. In the current investigation we intended to examine the variability of VEGFR1,2 and 3 among the experimental animals and human origin. We tend to investigate the degree of identity among human and animals and within animal groups. The VEGFR 1,2 and 3 amino acid sequences from human origin and from the murine origin (Mouse and Rat) were assessed for the homogeneity and difference in their protein confirmation. This will the give the information on the degree of the cross reactivity of the VEGFR antibodies against VEGFR 1,2,3 and species cross reactivity with murine origin.

Retrieval of sequences:-
The amino acid sequences of the surface marker Vascular Endothelial Growth Factor Receptor (VEGFR) was retrieved using NCBI and SWISS-PROT. Sequences of all three receptors, namely, VEGFR1, VEGFR2 and VEGFR3 from different species such as Homo sapiens (Human), Mus musculus (Mouse) and Rattus norvegicus (Rat) were used for this study. Established in 1988 as a national resource for molecular biology information, NCBI creates public databases, conducts research in computational biology, develops software tools for analyzing genome data, and disseminates biomedical information -all for the better understanding of molecular processes affecting human health and disease.
The Entrez system can provide views of gene and protein sequences and chromosome maps. Entrez can efficiently retrieve related sequences, structures, and references. Some textbooks are also available online through the Entrez system. Entrez Global Query is an integrated search and retrieval system that provides access to all databases simultaneously.
The sequences of the surface markers VEGFR 1, 2 and 3 were retrieved in FASTA format. VEGFR  Secondary structure prediction for proteins:-Secondary Structure Prediction was performed for the human VEGFR sequences using HNN -Hierarchical Neural Network method (Guermeur, 1997).Expression provides an interface to a large range of sophisticated secondary structure prediction algorithms. Protein Expression presently supports nine different secondary structure prediction algorithms. All computations are performed via the Network Protein Sequence Analysis server (PBIL, France). In this study, the prediction method followed was The HNN (Hierarchical Neural Network) prediction method.

Results and Discussion:-
Multiple Sequence Alignment of the retrieved sequences was obtained using DNAMAN Software. The VEGFR sequences within each class were compared for homology between the sequences from different origins.

84.60%
Phylogenetic Analysis:-The phylogeny between the VEGFR sequences retrieved from various sources was established by plotting trees and the results were obtained as follows.  The phylogenetic tree for VEGFR1 depicts that the sequences from Mus musculus (Mouse) and Rattus norvegicus (Rat) display greater similarity than with each of the sequences in comparison with Homo sapiens (Human) VEGFR 1 sequence. Similar results were obtained in case of phylogenetic tree generated between VEGFR2 as well as VEGFR 3 sequences of Homo sapiens (Human), Mus musculus (Mouse) and Rattus norvegicus (Rat).
Secondary structure prediction for human vegfr:-Secondary structure prediction was done for the Human VEGFR sequences using HNN Secondary Structure Prediction Method and the results were obtained as follows.
Date Set 1:-Human vegfr1: Hierarchical Neural Network result for: UNK_129930 Date Set 1. Using HNN for VEGFR 1 sequence, the length of the sequence was found to be 1338. The protein is chiefly folded into random coils (50.07%) and also consists of alpha helix (29.07%) and extended strand (20.85%). Data Set 2.Using HNN for VEGFR 2 sequence, the length of the sequence was found to be 1356. The protein is chiefly folded into random coils (52.06%) and also consists of alpha helix (24.48%) and extended strand (23.45%). 1331

Data Set 3. HUMAN VEGFR3: Hierarchical Neural Network result for: UNK_154230
Data Set 3. Using HNN for VEGFR sequence, the length of the sequence was found to be 1298. The protein is chiefly folded into random coils (51.00%) and also consists of alpha helix (29.58%) and extended strand (19.41%).

Discussion:-
Multiple sequence Alignment predicted the degree of variation of VEGFR 1 at genus levels:-Multiple sequence alignment (MSA) is an indispensable tool for studying the difference and homology of the macromolecules. There are many studies which compare with the protein sequence among different genus and species (Ramu Chenna et al., 2003). Therefore MSA becomes useful to know the difference in the amino acid composition which further gives the details of protein confirmation necessary for the functions like binding, activation etc. The current information of the VEGFR sequences retrieved from each species were assessed for homology using DNAMAN Software. The secondary protein structure further predicted showed the variations among each of the VEFG1, 2, and 3 subtypes. More over the results are mostly from consistency-based programs with higher accuracy determined the reliability of the results. TheVEGFR1 sequences showed an identity percentage of 87.58%between humans, mouse and rat (Table 1). On comparison of the individual VEGFR1 sequences among the species, we observed the homology between sequences from rat and mouse was 91.57% compared to the 80.92%homology between human and mouse and 81.12% homology between human and rat sequences respectively ( Table 2).

1332
In the case of VEGFR2 sequences, the indent percentage was found to be 90.14%. (Table 3)among human, mouse and rat. The individual comparison VEGFR2sequences homology between rat and mouse was 92.44%, compared to the 83.36%homology between human and mouse and 82.85% homology between human and rat sequences ( Table  4). The overall homology percentage for VEGFR 3 among the human, mouse and rat was 93.24% (Table 5). The sequence homology between rat and mouse is 95.78% compared to 85.46% human and mouse and 84.60% human and rat ( Table 6). The homology between the VEGFR 1, 2 and 3 sequences from mouse and rat were more homogenous compared to humans showing the higher percent of variation with highly evolved vertebrate (Higgins DG et al 1996).
Phylogenetic analysis of VEGFR 1,2 &3 reveals the evolutionary relationship between humans and murine species:-Phylogenetic trees were drawn using DNAMAN Software to establish the homology of the sequences from different sources. The algorithm and results were in compliance with the previous published works (Xin-Rong Liu et al., 2004) on evolutionary relationships and similarities among proteins.The phylogenetic tree for VEGFR1 depicts that the sequences from mouse and rat display greater similarity than with each of the sequences in comparison with humans (Figure1) Similar results were obtained in case of phylogenetic tree generated between VEGFR2 ( Figure  2) as well as VEGFR 3 ( Figure 3) sequences of human, mouse and rat (Hernández-García et al.,2015).
Secondary structure of VEFG prediction shows the variation on the protein confirmation across species:-Secondary Structure prediction for human VEGFR sequences using Hierarchical Neural Network Secondary Structure Prediction Method was on par with the previous studies (Guermeur Y, 1997, Chandrasekaran V et al., 2007 and the their proposed this algorithm for secondary structure for proteins.Using HNN for VEGFR 1 sequence, the length of the sequence was found to be 1338. The50.07% protein is chiefly folded into random coils with 29.07%of alpha helix and20.85% as extended strand (Data set 1).The length of the VEGFR 2 sequence was 1356. 52.06%protein is chiefly folded into random coils with 24.48% of alpha helix and 23,45% of extended strand (Data set 2). The length if the VEGFR 3 sequence was 1928. 51.00% protein is chiefly folded into random coils with 29.58% as alpha helix (29.58%) and 19.41% extended strand (Data set3). Thus, secondary structure prediction reveals the structural similarity between the classes of VEGFR sequences. All classes of VEGFRs reveal that large regions of their secondary structure consist of random coils and the remainder is folded into alpha helix (Wu G et al.,2013).

Conclusion:-
The MSCs VEGFR 1, 2 and 3 show homology and identity upon multiple sequence alignment. The sequences retrieved from Mus musculus (Mouse) and Rattus norvegicus (Rat) show greater homology in comparison with Homo sapiens (Human). The Phylogenetic Tree asserts the similarity and homology among VEGFR sequences from different species. Secondary structure prediction of the VEGFR sequences from Homo sapiens (Human) revealed that all the three receptors, i.e., VEGFR1, VEGFR2 and VEGFR3 show about 50% similarities in their secondary structure in random coils while remaining regions exist either as alpha helices or as extended strands.