In humans, the immunoglobulin super family containing leucine-rich repeat (ISLR) protein is encoded by the ISLR gene. Current RNA-seq studies show that the protein is highly expressed in the endometrium and ovary and shows expression among 25 other tissues. The protein is seen localized in the cytoplasm, plasma membrane, extracellular exosome, and platelet alpha granule lumen. Furthermore, the protein is known to play a role in platelet degranulation, cell adhesion, and response to elevated platelet cytosolic Ca2+.
Gene The aliases for ISLR are Meflin, HsT17563, and mesenchymal stromal-cell and fibroblast-expressing Linx paralogue. The gene is part of the I-set family.
The most updated annotation shows the gene spanning from 74,173,710 to 74,176,871 base pairs (3,161 bp) with location on the plus strand at position 15q24.1 (Chromosome 15). The gene contains 3 exons and 4 distinct introns.
Transcription
Known transcript variants The ISLR gene has two known transcript variants on the plus strand: ISLR transcript variant 1 and ISLR transcript variant 2. Both variants encode for the same protein.
Transcript variant 1 is the longer variant with a length of 2,331 bp and contains 2 exons.
Transcript variant 2 is the shorter variant with a length of 2,128 bp and contains 2 exons. This variant differs in the 5' UTR compared to variant 1.
Protein
Physical features
The human gene of ISLR has two alternatively spliced identical isoforms. The domains of the ISLR gene follows as: LRR_8 (leucine-rich repeat), LRR_RI (ribonuclease inhibitor), PCC (polycystin cation channel protein) Super family, and Ig (immunoglobulin).
The predicted isoelectric point of unmodified protein ISLR is 5.3. The calculated molar mass is 46.0 kDa.
The ISLR protein has 428 amino acids (aa) in humans. Through the Statistical Analysis of Protein Sequences (SAPS) tool, the percentage of most amino acid residues is about its average percentage among human proteins except leucine which shows high abundance compared to a normal protein. This is expected with the gene containing multiple LRR (leucine-rich repeats) structural motifs. There is a significantly low abundance of methionine (predicted to be 0.5%). In summary, the positively charged amino acid residues overcounts the negatively charged amino acid residues.
Through SAPS tool, there are two predicted identical four-block length repetitive structures that fall within LRR structural motifs: LSHL at 97-100 bp and 172-175 bp.
One high-scoring transmembrane segment was predicted through the SAPS tool from 411 to 428 aa (length 18) with a pocket from 417 to 418 aa. The Phobius prediction for the ISLR protein sequence illustrated the potential transmembrane domain and a signal peptide (Figure 2). The SignalP-5.0 prediction for the signal peptide reported a likelihood of 0.9989 with a cleavage site between position 18 and 19 with the probability of 0.9146.
Post-translational modifications Phosphorylation Sites
There are 31 predicted phosphorylation sites in the protein sequence for ISLR in humans from NetPhos. The results were filtered for best predictions for each residue display and accounted for serine, threonine, and tyrosine.
Through Eukaryotic Structural Motif (ELM tool) predictions, eight distinct phosphorylation sites were identified for the protein:
Glycogen synthase kinase-3 (GSK3) phosphorylation site at position 234-241 aa. Phosphatidylinositol 3-kinase related kinase (PIKK) phosphorylation site at position 238-244 aa. Casein kinase (CK1) phosphorylation site at position 335-341 aa. Two CK2 phosphorylation sites at positions 343-349 aa and 376-382 aa. Proline-Directed (MAPK) phosphorylation site at position 343-349 aa. Polo-like kinase phosphosites 1 (Plk1) at positions 336-342 aa and 351-357 aa. Ser/Thr residues are phosphorylated by the kinase. Plk4 phosphosite at position 415-421 aa. Ser/Thr residues are phosphorylated by the kinase. Two NEK2 phosphorylation sites at positions 415-420 bp and 423-428 aa. +Post-Translational Modifications predicted by Eukaryotic Structural Motif (ELM) Prediction in human ISLR proteinNamePosition (aa)Cell Compartment(s)N-degron1-3CytosolEndosome-Lysosome-Basolateral sorting signals2-7Cytosol, Endocytic vesicleNuclear Export Signal (NES)3-17Nucleus, cytosolPhosphotyrosine ligands bound by Src Homology 2 (SH2) domains238-241CytosolClass IV WW domain ligands343-348Cytosol, nucleusCyclin-dependent kinase subunit 1 (Cks1) ligand344-349Cytosol, nucleusTNFR-associated factors 6 (TRAF6) binding site345-353CytosolPeptide Amidation Site355-358Extracellular, secretory granuledi-Arg retention/retrieving signal357-359Endoplasmic reticulum membrane, ER-Golgi transport vesicle membrane, rough endoplasmic reticulum, endoplasmic reticulum cisterna, cytosol, integral proteinN-arginine dibasic (NRD) cleavage site357-359Extracellular, Golgi apparatus, cell surfaceSrc Homology 3 (SH3) ligand375-381Plasma membrane, focal adhesion, cytosolGlycosaminoglycan attachment site379-381Extracellular, Golgi apparatusSTAT5 SH2 domain binding motif370-373 389-392CytosolPDZ domain ligands423-428Cytosol, internal side of plasma membranePex14 ligand motif424-428Cytosol, peroxisome, glycosome Table 1. Results of ELM motif search after context, structural, and globular domain filtering with acceptable structural score (above medium threshold score). There are 23 total identified post-translational modifications including phosphorylation sites in the human protein of ISLR.
Glycosylation Sites
Through the Simple Modular Architecture Research (SMART) tool in Figure 3, an annotation predicted three N-linked glycosylation sites (red circles, starting from the left): 51 aa, 60 aa, and 309 aa. The LRR structural motifs and immunoglobulin C-2 type (IGc2) domain are shown in the diagram (Figure 3).
Amidation Sites
Predicted with MyHits which investigates relationships between protein sequences and motifs, an amidation site motif was confidently predicted at position 355-358 aa.
Palmitoylation sites
Three palmitoylation sites were predicted (Table 2). +Predicted Palmitoylation sites in CSS-Palm for the ISLR human protein PositionPeptideScoreCutoff19LLGLAQACPEPCDCG21.1943.71723AQACPEPCDCGEKYG13.2210.72225ACPEPCDCGEKYGFQ4.4633.717 Table 2. Results of the CSS-Palm for the human protein of ISLR.
GPI-Modifications
Predicted with big-PI predictor, one glucose phosphate isomerase (GPI) modification was found at position 401 aa (best site) with P-value score of 1.71e-03.
Secondary and Tertiary Structure Of all the predicted beta sheets, four stretches at 253-260 aa, 265-272 aa, 323-331 aa, and 335-346 aa were identified with high confidence using CFSSP and Phyre2. Of all the predicted alpha helices, three alpha helices at 5-15 aa, 189-195 aa, and 214-216 aa were identified with high confidence as well. A tertiary model of the human ISLR protein predicted by I-TASSER shows a combination of some alpha helices and beta sheets (Figure 1). Based on the secondary structure prediction of the protein in I-TASSER, the locations of the four beta sheets and three alpha helices confirms the predictions of high confidence made by CFSSP and Phyre2.
Subcellular Localization The ISLR protein in humans is expected to localize throughout a cell, including extracellular region, based on the predicted results of PSORT II. The Reinhardt's method for cytoplasmic/nuclear discrimination predicted the protein to be more cytoplasmic with a reliability of 76.7. Additionally, ISLR was shown to localize in the cytoplasm based on the polyclonal antibody results in immunohistochemically stained human tissues in myocytes, glandular cells, skin, hepatocytes. Immunofluorescent staining of ISLR in human cell line BJ (fibroblasts) showed localization to the plasma membrane using ISLR Polyclonal Antibody as well.
Expression In humans, RNA-seq was conducted on tissue samples from 95 individuals representing 27 different tissues to determine tissue-specificity of all protein-coding genes. Notably, there is high expression of ISLR in endometrium and ovary and visible expression among 25 other tissues. Another study of RNA Sequencing of total RNA from 20 human tissues demonstrated high expression of ISLR in uterus. Tissue-specific circular RNA induction during human fetal development showed steady expression of ISLR throughout the development with a high increase at 10 weeks for stomach. Expression remained notably high to 20 weeks for stomach.
In the annotated figure, an in situ Hybridization on a 56-days old male mouse brain (sagittal cut) demonstrated expression in the olfactory areas and hippocampal formation (Figure 4).
Based on Protein Abundance Database (PAXdb 4.1), the human protein of ISLR is shown with high protein abundance (ppm value > 1) relative to the whole organism.
Expression in Clinical Studies Expression profiling by microarray of ISLR in female human subjects demonstrated overexpression of ISLR in breast lipotransfer white adipose tissue CD34+ cells and significantly lower expression in leukapheresis CD34+ cells.
Expression profiling by microarray of ISLR in human subjects demonstrated overexpression in non-union skeletal fractures compared to low expression in normal fractures.
Expression profiling by microarray of ISLR in obese female human subjects demonstrated consistent low expression of ISLR in subjects that followed a short-term low-fat hypocaloric diet.
Regulation of Expression
Gene level regulation There is one promoter region in the ISLR gene with a predicted length of 1,912 bp (Figure 5) extracted from Genomatix. Additionally, there is a polyadenylation signal at 3,142 bp in the ISLR nucleotide sequence (humans).
There are six distinct transcription factors that bind onto the promoter region of ISLR from Genomatix predictions: two SMAD factors, sine oculis homeobox (SIX), heat shock factor (HSF), PRDM, Snail, and cell cycles gene homology region (CHR).
Genomatix results predicted more transcription factor binding sites in ISLR with the highest matrix similarity (0.97~0.99) such as:
Myeloid zinc finger 1 factor (MZF1) Nuclear factor of activated T-cells Hepatocyte nuclear factor 3 (alpha, beta) (FOXA1, FOXA2) E2F transcription factor 1 Eomesodermin, TBR-2 (T-box, brain, 2) (Brachyury gene, mesoderm developmental factor) Homeodomain factor Nkx-2.5/Csx Estrogen-related receptor alpha (secondary DNA binding preference) Early B-cell factor 1 (Neuron specific olfactory factor) Nascent polypeptide-associated complex subunit alpha 1 IKAROS family zinc finger 3 (Aiolos) 3' half site of ZTRE motif (zinc transcriptional regulatory element) AREB6 (Atp1a1 regulatory element binding factor 6)
Transcript level regulation The human gene of ISLR is predicted to be targeted by 85 miRNAs in miRDB. The top scoring (>88) miRNAs are has-miR-5197-3p, has-miR-4688, has-miR-3150a-3p, has-miR-16-5p, has-miR-195-5p, has-miR-15a-5p, and has-miR-6763-5p.
RBPmap, which maps predicted binding sites of RNA binding proteins, showed multiple conserved motifs in evolution relative to the human ISLR mRNA transcript variant 1 sequence such as:
BRUNOL6 CPEB2 and CPEB4 ESRP2 FMR1 FUS FXR1 and FXR2 G3BP2 HNRNPA1, HNRNPA1L2, HNRNPA2B1, HNRNPC, HNRNPCL1, HNRNPF, HNRNPH1, HNRNPH2, HNRNPK, HNRNPL, HNRNPM, HNRNPU, HNRPLL. HuR IGF2BP2 and IGF2BP3 KHDRBS2 LIN28A
Homology and Evolutionary History
Paralogs Currently, there is one other paralog in humans known as ISLR 2 and two paralogous domains: LRRN4 (protein precursor 4) and LRRN4CL (protein precursor 4 C-terminal like).
Orthologs/Distant Homologs