Splice Variant of Spalax Heparanase Skipping Exon 12

The subterranean blind mole rat, Spalax, has evolved significantly over 47 million years to thrive in its underground habitat. A key enzyme in this adaptation is heparanase, which degrades heparan sulfate (HS) in the extracellular matrix (ECM), facilitating angiogenesis and releasing growth factors for endothelial cells. Spalax heparanase has various splice variants influencing tumor growth and metastasis differently. We report a novel splice variant from a hypoxia-exposed kidney sample resulting from exon 12 skipping. This variant maintains the translation frame but lacks enzymatic activity, offering insights into Spalax’s unique adaptations.


Introduction
The subterranean blind mole rat (Spalax) is a fascinating mammal that spends the majority of its life living underground.Over 47 million years, Spalax has evolved unique adaptations to its subterranean environment [1].One notable adaptation is the atrophy of its eyes, as living in darkness in subterranean tunnels made sight less essential [2].Despite this, Spalax is still able to distinguish between light and darkness [3].Spalax's limbs have also become shorter to accommodate the narrow tunnels [2].However, Spalax's muscles are much more developed and have a higher density of blood vessels compared with other rodents [4].In addition, Spalax has developed several extraordinary abilities to navigate and communicate underground.It can perceive and utilize the Earth's magnetic field to orient itself in space while digging underground [5,6].Furthermore, Spalax has developed seismic communication using low-frequency and patterned substrate-borne vibrations generated by head thumping [7].Spalax lives much longer than rats and mice, reaching up to 20 years.This remarkable longevity is potentially supported by the damage-free shortening of telomeres [8].These unique adaptations have allowed Spalax to thrive in its subterranean habitat and provide valuable insights into how species adapt to extreme environments.
In Israel, the genus Spalax is part of the superspecies Spalax ehrenbergi [9].Four distinct Israeli species share similar morphology but differ in their diploid chromosome numbers.Specifically, Spalax galili, S. golani, S. carmeli, and S. judaei have 52, 54, 58, and 60 chromosomes, respectively [9].One unique characteristic of Spalax is its ability to maintain high levels of metabolic activity in both hypoxic and hypercapnic conditions [10].Moreover, Spalax has a lower coronary artery resistance in its heart compared with other rodents [11].In terms of its lipid profile, Spalax has high levels of high-density lipoproteins (HDLs) and low levels of low-density lipoproteins (LDLs) and triglycerides as compared to humans [12].Additionally, Spalax has higher antioxidant serum content than both humans and mice [12].
In our previous study, we reported on the successful cloning of Spalax heparanase and its ability to degrade heparan sulfate [33].We also identified multiple splice variants of heparanase in Spalax.For example, splice 7 results from the skipping of exon 7, while splice 36 is generated by skipping parts of exons 3 and 6 and all of exons 4 and 5. Splice 36 functions as a dominant negative to wild-type heparanase, thereby suppressing HS degradation, tumor growth, and metastasis [34].In addition, splice 67 arises from the skipping of exons 6 and 7, and splice 612 from skipping exons 6 to 12.These two splice variants generate truncated heparanase proteins that share the same N-terminus as the native wild-type enzyme, but each possesses a unique C-terminus [35].In the current study, we describe a new splice variant, splice 12 of Spalax heparanase, generated by the skipping of exon 12 and is devoid of heparanase enzymatic activity.

Animals
The Spalax used in this study were captured from their natural habitat by removing the dirt covering their tunnel openings.Once the animals were detected near the entrance trying to re-block it with dirt, the hunters blocked their way back and captured them.Since Spalax do not reproduce in captivity, maintaining wild-caught animals is essential for research.The captured Spalax were then housed individually at the animal facility of the Institute of Evolution, University of Haifa, Israel.They were maintained under controlled conditions at 22-24 • C and fed a diet of carrots and apples.The study utilized adult Spalax judaei from the Anza population, weighing between 100 to 150 g.The animals were exposed to a hypoxic pulse (6% O 2 for 3 h) and subsequently euthanized by injection of Ketaset CIII (Fort Dodge Animal Health, Fort Dodge, IA, USA) at a dose of 5 mg/kg body weight.Whole organs were removed and immediately frozen in liquid nitrogen.

RNA and cDNA Preparation
Total RNA was meticulously extracted from tissues using TRI Reagent (Molecular Research Center, Cincinnati, OH, USA) according to the manufacturer's detailed instructions.Subsequently, cDNA was synthesized by reverse transcription of 1 µg of total RNA with Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI, USA), utilizing oligo(dT)15 and random primers to ensure comprehensive coverage of mRNA transcripts [33,36].

Cloning of Spalax Heparanase Splice Variants
To clone Spalax heparanase splice variants, cDNAs were prepared from kidney tissues, and Spalax-specific primers were used [33].To clone splice 12, forward primer Mf: GGTCAACCTCGAGGAAAGACAGTTAA and reverse primer s3 ′ Lb: TCATAGA-CAAGCAGCAACTTTGGCATTTC (Sigma Genosys, Rehovot, Israel) were utilized.PCR was performed using TaqDNA polymerase (Qbiogene, Illkirch, France) and kidney cDNA as a template.The presence of splice 12 was confirmed by PCR utilizing sHep1742f and s3 ′ Lb primers.The amplified band was sub-cloned into the pGEM-Teasy vector, sequenced with gene-and vector-specific primers using an automated DNA sequencer (ABI Prism model 310 Genetic Analyzer; Perkin Elmer, Foster City, CA, USA), and then constructed into the pcDNA3 expression vector (Invitrogen, Leek, The Netherlands) containing the fulllength Spalax heparanase by means of digestion and ligation with site-specific restriction enzymes and T4 ligase (Promega, Madison, WI, USA).

Cells and Transfections
Uppsala 87 Malignant Glioma (U87) cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 4.5 g/L glucose, 10% fetal calf serum (FCS), and antibiotics, as described [34].Cells were grown in 60 mm tissue culture dishes and transfected with a total of 1-2 µg of plasmid DNA mixed with 6 µL of Fu Gene transfection reagent (Roche Applied Science, Mannheim, Germany) and 94 µL of DMEM.Transiently transfected cells were obtained after 24-48 h of incubation at 37 • C. Stable populations of transfected cells were selected with G418 (Sigma Genosys, Rehovot, Israel).
We chose U87 cells for transfections and heparanase enzymatic activity assays because these cells do not have endogenous expression of heparanase.This lack of endogenous heparanase ensures that any observed enzymatic activity or effects on cell behavior can be attributed solely to the transfected heparanase constructs, thereby providing a clear and uncontaminated assessment of the splice variants' functional properties.This makes U87 cells an ideal model system for studying the specific activities of heparanase splice variants.

Cloning of Splice Variant #12 of Spalax Heparanase
Splice variant #12 of Spalax heparanase was cloned from a kidney of Spalax judaei of the Anza population that was exposed to a 3 h pulse of hypoxia with 6% O 2 .This variant is different from the wild-type heparanase and other splice variants, as it results from splicing out of exon 12 [39] (nucleotides 1770-1916) (Figure 1).As a result, the cDNA of splice variant #12 is 147 base pairs shorter than the wild-type sequence, but it does not cause any frameshift.PCR products were amplified using kidney cDNA as a template and primers designed around the exon 12 segment.Gel electrophoresis of these products revealed both the wild-type and spliced forms (Figure 2A, lane 2).
is different from the wild-type heparanase and other splice variants, as it results from splicing out of exon 12 [39] (nucleotides 1770-1916) (Figure 1).As a result, the cDNA of splice variant #12 is 147 base pairs shorter than the wild-type sequence, but it does not cause any frameshift.PCR products were amplified using kidney cDNA as a template and primers designed around the exon 12 segment.Gel electrophoresis of these products revealed both the wild-type and spliced forms (Figure 2A, lane 2).

Expression of Spalax Heparanase Splice Variants in U87 Cells
Splice variant 12 of Spalax heparanase had a shorter amino acid sequence, lacking 49 amino acids compared with the wild-type protein.The splicing out of exon 12 results in a substitution of Lys473 with Gln at the junction of exons 11 and 13 (Figure 3).To examine the expression and secretion of different heparanase splice variants, U87 cells were is different from the wild-type heparanase and other splice variants, as it results from splicing out of exon 12 [39] (nucleotides 1770-1916) (Figure 1).As a result, the cDNA of splice variant #12 is 147 base pairs shorter than the wild-type sequence, but it does not cause any frameshift.PCR products were amplified using kidney cDNA as a template and primers designed around the exon 12 segment.Gel electrophoresis of these products revealed both the wild-type and spliced forms (Figure 2A, lane 2).

Expression of Spalax Heparanase Splice Variants in U87 Cells
Splice variant 12 of Spalax heparanase had a shorter amino acid sequence, lacking 49 amino acids compared with the wild-type protein.The splicing out of exon 12 results in a substitution of Lys473 with Gln at the junction of exons 11 and 13 (Figure 3).To examine the expression and secretion of different heparanase splice variants, U87 cells were

Expression of Spalax Heparanase Splice Variants in U87 Cells
Splice variant 12 of Spalax heparanase had a shorter amino acid sequence, lacking 49 amino acids compared with the wild-type protein.The splicing out of exon 12 results in a substitution of Lys473 with Gln at the junction of exons 11 and 13 (Figure 3).To examine the expression and secretion of different heparanase splice variants, U87 cells were transfected with plasmids containing the Mock empty vector, wild-type (WT), splice 7 (S7), splice 12 (S12), or splice 36 (S36) of Spalax heparanase.Western blot analysis was performed using polyclonal anti-heparanase antibodies #1453 to detect heparanase in the conditioned media and cell lysates (Figure 2B).The results showed that only Spalax WT heparanase was detected in the conditioned media in its latent form, while the cell lysates contained both the latent and active forms of the protein.The splice variants S7, S12, and S36 were expressed as a single band in the cell lysates, whereas the wild-type Spalax heparanase was expressed as two bands, representing its latent and active forms.(Figure 2B).performed using polyclonal anti-heparanase antibodies #1453 to detect heparanase in conditioned media and cell lysates (Figure 2B).The results showed that only Spalax heparanase was detected in the conditioned media in its latent form, while the cell lys contained both the latent and active forms of the protein.The splice variants S7, S12, S36 were expressed as a single band in the cell lysates, whereas the wild-type Spalax h aranase was expressed as two bands, representing its latent and active forms.(Figure

Heparanase Enzymatic Activity
U87 cells were transfected with either an empty plasmid (M), plasmids containing wild-type (WT) Spalax heparanase, splice variants 7 (S7), 12 (S12), or 36 (S36).Cell lysates were incubated with naturally produced, sulfate-labeled extracellular matrix (ECM) for 4 h at 37 • C (pH 6.0).Degradation fragments released into the medium were then analyzed by gel filtration on Sepharose 6B.Results showed that only the lysates of cells transfected with WT Spalax heparanase were able to release low-molecular-weight-labeled degradation fragments of HS from the ECM.In contrast, lysates of cells transfected with S7, S12, or S36 Spalax heparanase or Mock empty plasmid (M) did not result in degradation of HS (Figure 4).

Heparanase Enzymatic Activity
U87 cells were transfected with either an empty plasmid (M), plasmids containing wild-type (WT) Spalax heparanase, splice variants 7 (S7), 12 (S12), or 36 (S36).Cell lysates were incubated with naturally produced, sulfate-labeled extracellular matrix (ECM) for 4 h at 37 °C (pH 6.0).Degradation fragments released into the medium were then analyzed by gel filtration on Sepharose 6B.Results showed that only the lysates of cells transfected with WT Spalax heparanase were able to release low-molecular-weight-labeled degradation fragments of HS from the ECM.In contrast, lysates of cells transfected with S7, S12, or S36 Spalax heparanase or Mock empty plasmid (M) did not result in degradation of HS (Figure 4).

Discussion
Heparanase is an enzyme that specifically degrades heparan sulfate [16,36].It is highly expressed in platelets, and upon activation at sites of injury, the secreted enzyme degrades the endogenous heparin/HS found in the blood, thereby promoting blood clotting [16].In addition to its roles in platelet function and wound healing, heparanase has been implicated in various pathological processes, including inflammation, angiogenesis, metastasis, and tumor growth [16,17].Heparanase expression has been reported in various cell types, including leukocytes, endothelial cells, and cancer cells [17,21,[23][24][25]37].In cancer, heparanase promotes tumor growth and metastasis by facilitating tumor cell invasion and angiogenesis through degradation of the extracellular matrix and the release of pro-angiogenic and growth-promoting factors [15,17].Heparanase inhibitors have been developed as potential anti-cancer agents, and clinical trials are currently underway to evaluate their efficacy [40,41].Overall, heparanase is a multifaceted enzyme that plays important roles in various physiological and pathological processes, and its regulation and therapeutic targeting have significant implications for human health.

Discussion
Heparanase is an enzyme that specifically degrades heparan sulfate [16,36].It is highly expressed in platelets, and upon activation at sites of injury, the secreted enzyme degrades the endogenous heparin/HS found in the blood, thereby promoting blood clotting [16].In addition to its roles in platelet function and wound healing, heparanase has been implicated in various pathological processes, including inflammation, angiogenesis, metastasis, and tumor growth [16,17].Heparanase expression has been reported in various cell types, including leukocytes, endothelial cells, and cancer cells [17,21,[23][24][25]37].In cancer, heparanase promotes tumor growth and metastasis by facilitating tumor cell invasion and angiogenesis through degradation of the extracellular matrix and the release of pro-angiogenic and growth-promoting factors [15,17].Heparanase inhibitors have been developed as potential anti-cancer agents, and clinical trials are currently underway to evaluate their efficacy [40,41].Overall, heparanase is a multifaceted enzyme that plays important roles in various physiological and pathological processes, and its regulation and therapeutic targeting have significant implications for human health.
We previously reported the expression of wild-type heparanase in Spalax kidney, highlighting the critical role of this organ in maintaining homeostasis and adapting to hypoxic conditions [33].The kidneys' functions in blood filtration and oxygen regulation make them an ideal model for studying physiological responses to hypoxia [42,43].During our investigations of heparanase activity in various kidney tissues of Spalax, we identified a novel splice variant, S12.This discovery underscores the unique genetic adaptations of Spalax to its subterranean habitat and emphasizes the significance of heparanase splice variants in these processes.
Transfecting U87 cells with Spalax heparanase splice variants S7, S12, or S36 results in specific functional changes: S7 increases tumor growth despite lacking enzymatic activity [34]; S12, reported for the first time in this article, lacks enzymatic activity, and its effect on cell behavior is not fully characterized yet; and S36 reduces tumor growth by inhibiting heparan sulfate degradation [34].This highlights the diverse functional roles of heparanase splice variants and their potential implications in tumor biology [15].
The cloning of splice variant 12 of Spalax heparanase provides an opportunity for further research into the function and role of heparanase in health and disease.Studying the splice variants of heparanase can enhance our understanding of the protein's diversity and its potential roles in various cellular processes.Furthermore, the ability to express and study different splice variants of heparanase in vitro can provide insights into their differential activities, which may have implications for developing targeted therapeutics for cancer, inflammation, kidney dysfunction, viral infections, and other pathologies.
Spalax underwent genetic adaptations to survive the harsh underground environment.One notable adaptation is the evolution of a unique form of hemoglobin, which has a higher affinity for oxygen than the hemoglobin found in other mammals.This adaptation allows Spalax to extract oxygen more efficiently from the low-oxygen environment in its burrow [4,44,45].Additionally, Spalax has adaptations in its metabolism, such as a lower metabolic rate, which allow it to conserve energy and survive periods of food scarcity [2,44,46].Spalax also has an enhanced immune system, likely due to the high pathogenicity in its underground environment [1,9,47].These adaptations in Spalax have led to interest in studying the molecular mechanisms underlying its unique physiology and potential applications for human health.
Alternative splicing can result in the production of multiple mRNA variants of the same gene, leading to different protein isoforms with varying functions or properties [48,49].Exon skipping, partial skipping of an exon, or intron retention can all contribute to alternative splicing, and the resulting protein isoforms likely have a different amino acid sequence and structure as compared with the original protein [34,49].When the skipped nucleotide number is not a multiple of three, a frameshift occurs, and the resulting protein has a unique C-terminal sequence [35].This can affect the protein's enzymatic activity, cellular localization, or interactions with other molecules [15,[34][35][36].
Splice #12 variant results from the splicing out of exon 12, which causes a deletion of 147 base pairs in the cDNA sequence.This deletion maintains the reading frame and results in a protein that is shorter by 49 amino acids compared with the wild-type heparanase.This deletion affects the C-terminus of the protein, which contains functional domains important for heparanase secretion, structural integrity, enzymatic and non-enzymatic activities, interaction with other proteins, and pro-tumorigenic properties [50][51][52][53][54].
The discovery of splice variant 12 of Spalax heparanase provides an opportunity to further explore the diversity and potential roles of heparanase in various cellular processes, including the adaptation of Spalax to its harsh underground environment.Further research into heparanase and its splice variants can provide valuable insights into its regulation and therapeutic targeting for various diseases [55].

Figure 1 .
Figure 1.Schematic drawing of Spalax wild-type (WT) heparanase and its splice variants.Splice 7 (S7), splice 12 (S12) and splice 36 (S36), results of skipping of nucleotides numbers that are a multiple of three, and maintain the reading frame.Splice 67 (S67) and splice 612 (S612) skips nucleotide numbers that are not a multiple of three, resulting in a frameshift, and proteins that has unique Cterminals sequences.

Figure 2 .
Figure 2. (A) Cloning of splice variant #12 of Spalax heparanase.Semi-quantitative RT-PCR using Spalax-specific primers located around the heparanase cDNA region encoded by exon 12. Bands of 336 bp represent the wild-type enzyme, while those of 189 bp represent its splice 12 form.Lane 1: reaction mixture alone; Lane 2: cDNA of kidney; Lanes 3 and 4, plasmids containing the cDNA sequence of the wild-type Spalax heparanase and the splice 12 variant, respectively.(B) Western blot analysis.U87 cells transfected with Mock empty plasmid, or plasmids containing wild-type (WT), splice 7 (S7), splice 12 (S12), or splice 36 (S36) of Spalax heparanase were cultured, as described in Methods.Western blot analysis of conditioned media (left) and cell lysates (right) was performed using polyclonal anti-heparanase antibodies #1453.

Figure 1 .
Figure 1.Schematic drawing of Spalax wild-type (WT) heparanase and its splice variants.Splice 7 (S7), splice 12 (S12) and splice 36 (S36), results of skipping of nucleotides numbers that are a multiple of three, and maintain the reading frame.Splice 67 (S67) and splice 612 (S612) skips nucleotide numbers that are not a multiple of three, resulting in a frameshift, and proteins that has unique C-terminals sequences.

Figure 1 .
Figure 1.Schematic drawing of Spalax wild-type (WT) heparanase and its splice variants.Splice 7 (S7), splice 12 (S12) and splice 36 (S36), results of skipping of nucleotides numbers that are a multiple of three, and maintain the reading frame.Splice 67 (S67) and splice 612 (S612) skips nucleotide numbers that are not a multiple of three, resulting in a frameshift, and proteins that has unique Cterminals sequences.

Figure 2 .
Figure 2. (A) Cloning of splice variant #12 of Spalax heparanase.Semi-quantitative RT-PCR using Spalax-specific primers located around the heparanase cDNA region encoded by exon 12. Bands of 336 bp represent the wild-type enzyme, while those of 189 bp represent its splice 12 form.Lane 1: reaction mixture alone; Lane 2: cDNA of kidney; Lanes 3 and 4, plasmids containing the cDNA sequence of the wild-type Spalax heparanase and the splice 12 variant, respectively.(B) Western blot analysis.U87 cells transfected with Mock empty plasmid, or plasmids containing wild-type (WT), splice 7 (S7), splice 12 (S12), or splice 36 (S36) of Spalax heparanase were cultured, as described in Methods.Western blot analysis of conditioned media (left) and cell lysates (right) was performed using polyclonal anti-heparanase antibodies #1453.

Figure 2 .
Figure 2. (A) Cloning of splice variant #12 of Spalax heparanase.Semi-quantitative RT-PCR using Spalax-specific primers located around the heparanase cDNA region encoded by exon 12. Bands of 336 bp represent the wild-type enzyme, while those of 189 bp represent its splice 12 form.Lane 1: reaction mixture alone; Lane 2: cDNA of kidney; Lanes 3 and 4, plasmids containing the cDNA sequence of the wild-type Spalax heparanase and the splice 12 variant, respectively.(B) Western blot analysis.U87 cells transfected with Mock empty plasmid, or plasmids containing wild-type (WT), splice 7 (S7), splice 12 (S12), or splice 36 (S36) of Spalax heparanase were cultured, as described in Methods.Western blot analysis of conditioned media (left) and cell lysates (right) was performed using polyclonal anti-heparanase antibodies #1453.

Figure 3 .
Figure 3. Amino acid sequences of splice 12 (top) and wild-type (bottom) Spalax judaei heparanase.Splice 12 lacks 49 amino acids compared with the wild-type heparanase enzyme-represent amino acids missing in splice 12: represents conserved substitutions (amino acids with similar properties), and * represent a stop codon.
patent number: US8093031.US Provisional application number: US60/718354.Patent assigned to Carmel Haifa University Economic Corp Ltd.(Haifa, Israel).Author Contributions: Conceptualization, N.J.N., A.A. and E.N.; methodology, N.J.N., A.A. and E.N.; software, N.J.N. and A.A.; formal analysis, N.J.N., A.A. and E.N.; writing-original draft preparation, N.J.N. and A.A.; writing-review and editing, N.J.N., A.A. and E.N.All authors have read and agreed to the published version of the manuscript.Funding: This research received no external funding.Institutional Review Board Statement: The experiments were conducted in accordance with protocols approved by the Ethics Committee of the University of Haifa.Informed Consent Statement: Not applicable.Data Availability Statement: All sequences cloned were published under the US patent US8093031, available through Google Patents: https://patentimages.storage.googleapis.com/7f/a6/90/922bf795478f24/US8093031.pdf(accessed 6 August 2024).