Heparin-binding Activity Research Articles

External Trigger Free Charge Switchable Cationic Ligands in the Design of Safe and Effective Universal Heparin Antidote.

Thrombosis, the formation of blood clots within a blood vessel, can lead to severe complications including pulmonary embolism, cardiac arrest, and stroke. The most widely administered class of anticoagulants is heparin-based anticoagulants such as unfractionated heparin, low-molecular weight heparins (LMWHs), and fondaparinux. Protamine is the only FDA-approved heparin antidote. Protamine has limited efficacy neutralizing LMWHs and no reversal activity against fondaparinux. The use of protamine can lead to complications, including excessive bleeding, hypotension, and hypersensitivity, and has narrow therapeutic window. In this work, a new concept in the design of a universal heparin antidote: switchable protonation of cationic ligands, is presented. A library of macromolecular polyanion inhibitors (MPIs) is synthesized and screened to identify molecules that can neutralize all heparins with high selectivity and reduced toxicity. MPIs are developed by assembling cationic binding groups possessing switchable protonation states onto a polymer scaffold. By strategically selecting the identity and modulating the density of cationic binding groups on the polymer scaffold, a superior universal heparin reversal agent is developed with improved heparin-binding activity and increased hemocompatibility profiles leading to minimal effect on hemostasis. The activity of this heparin antidote is demonstrated using in vitro and in vivo studies.

Crystal structure of the C-terminal domain of envelope protein VP37 from white spot syndrome virus reveals sulphate binding sites responsible for heparin binding.

White spot syndrome virus (WSSV) is the most virulent pathogen causing high mortality and economic loss in shrimp aquaculture and various crustaceans. Therefore, the understanding of molecular mechanisms of WSSV infection is important to develop effective therapeutics to control the spread of this viral disease. In a previous study, we found that VP37 could bind with shrimp haemocytes through the interaction between its C-terminal domain and heparin-like molecules on the shrimp cells, and this interaction can also be inhibited by sulphated galactan. In this study, we present the crystal structure of C-terminal domain of VP37 from WSSV at a resolution of 2.51 Å. The crystal structure contains an eight-stranded β-barrel fold with an antiparallel arrangement and reveals a trimeric assembly. Moreover, there are two sulphate binding sites found in the position corresponding to R213 and K257. In order to determine whether these sulphate binding sites are involved in binding of VP37 to heparin, mutagenesis was performed to replace these residues with alanine (R213A and K257A), and the Surface Plasmon Resonance (SPR) system was used to study the interaction of each mutated VP37 with heparin. The results showed that mutants R213A and K257A exhibited a significant loss in heparin binding activity. These findings indicated that the sites of R213 and K257 on the C-terminal domain of envelope protein VP37 are essential for binding to sulphate molecules of heparin. This study provides further insight into the structure of C-terminal domain of VP37 and it is anticipated that the structure of VP37 might be used as a guideline for development of antivirus agent targeting on the VP37 protein.

BMP6 binding to heparin and heparan sulfate is mediated by N-terminal and C-terminal clustered basic residues

BackgroundThe bone morphogenetic protein 6 (BMP6) is a crucial inducer of hepcidin, the peptide hormone that regulates the iron availability in our body. Hepcidin expression is influenced by hepatic heparan sulfate (HS) and by heparin administration, suggesting BMP6 interaction with heparin/HS. The BMP2/4 subfamily has been deeply characterized to have a N-terminal heparin/HS binding domain (HBD), whose basic residues contact the sulfate groups on heparin and HS. Such detailed characterization is still required for other, structurally different BMPs, including BMP6. MethodsBMP6 peptides encompassing potential HBDs were analysed on heparin-functionalized plates and microcantilevers, and on membrane HS expressing CHO-K1 cells. Monomeric wild-type BMP6 and mutants were produced, substituting the basic residues with non-charged ones, and their affinity to the heparin-column was measured. The BMP6-heparin interaction was also predicted at atomic level by in silico molecular dynamics. ResultsN-terminal and C-terminal BMP6 peptides showed high heparin affinity in solid-phase assays. The mutation of the two sites (R5L, R6S, R7L and K126N, K127N, R129S) abolished the heparin-binding activity of the recombinant monomeric BMP6. Monomeric BMP6 and peptides specifically bound to membrane HS of CHO-K1 cells through the same domains. Molecular dynamic studies supported the role of the two HBDs, suggesting a cooperative behaviour. ConclusionsIn BMP6, N-terminal (R5, R6, R7) and C-terminal (K126, K127, R129) domains mediate the interaction with heparin and HS. General significanceThis study provides the molecular mechanism supporting the use of heparin to sequester BMP6 and inhibit hepcidin expression, a novel clinical approach for high-hepcidin iron disorders.

Sub-fertility in crossbred bulls: deciphering testicular level transcriptomic alterations between zebu (Bos indicus) and crossbred (Bos taurus x Bos indicus) bulls

BackgroundThe incidence of poor semen quality and sub-fertility/infertility is higher in crossbred as compared to Zebu males. Several attempts have been made to understand the possible reasons for higher incidence of fertility problems in crossbred males, at sperm phenotype, proteome and genome level but with variable results. Since the quality of the ejaculated spermatozoa is determined by the testicular environment, assessing the testicular transcriptome between these breeds would help in identifying the possible mechanisms associated with infertility in crossbred bulls. However, such information is not available. We performed global transcriptomic profiling of testicular tissue from crossbred and Zebu bulls using Agilent Bos taurus GXP 8X60k AMADID: 29411 array. To the best of our knowledge, this is the first study comparing the testicular mRNAs between crossbred and Zebu bulls.ResultsOut of the 14,419 transcripts detected in bovine testis, 1466 were differentially expressed between crossbred and Zebu bulls, in which 1038 were upregulated and 428 were downregulated in crossbred bulls. PI4KB and DPY19L2 genes, reported to be involved in sperm capacitation and acrosome formation respectively, were among the top 10 downregulated transcripts in crossbred testis. Genes involved in ubiquitination and proteolysis were upregulated, while genes involved in cell proliferation, stem cell differentiation, stem cell population maintenance, steroidogenesis, WNT signalling, protein localization to plasma membrane, endocannabinoid signalling, heparin binding, cAMP metabolism and GABA receptor activity were downregulated in crossbred testis. Among the 10 genes validated using qPCR, expression of CCNYL, SOX2, MSMB, SPATA7, TNP1, TNP2 and CRISP2 followed the same trend as observed in microarray analysis with SPATA7 being significantly downregulated and transition proteins (TNP1, TNP2) being significantly upregulated in crossbred bulls.ConclusionsAbundant proteolysis by ubiquitination and downregulation of WNT signaling, cell proliferation, differentiation and steroidogenesis might be associated with higher incidence of poor semen quality and/or sub-fertility/infertility in crossbred bulls as compared to Zebu bulls. Downregulation of SPATA7 (Spermatogenesis Associated 7) and upregulation of transition proteins (TNP1 and TNP2) in crossbred bull testis might be associated with impaired spermatogenesis processes including improper chromatin compaction in crossbred bulls.

Identification of important invasion and proliferation related genes in adrenocortical carcinoma.

Adrenocortical carcinoma (ACC) is an end-stage hormonal syndrome. Although profound attempts have been made to illuminate the pathogenesis, the molecular mechanisms of ACC remain to be clarified. To identify the important genes in the progression of ACC, microarray datasets GSE19775 was downloaded from the gene expression omnibus database. The differentially-expressed genes (DEGs) were identified, and pathway and GO enrichment analyses were performed. The protein-protein interaction (PPI) network was constructed and the module analysis was performed using the protein interaction network analysis and Cytoscape. Also constructed target genes-miRNA regulatory network and target genes-TF regulatory network. Correlation of the hub genes were analyzed in The Cancer Genome Atlas. The prognostic values of hub genes were further validated by online tool UALCAN. Mutation analysis was done by online tool CBio Cancer Genomics Portal. A total of 884 DEGs were identified, with 441 in up regulation and 443 in down regulation. Pathways in catecholamine biosynthesis, aldosterone synthesis and secretion, pyrimidine deoxyribonucleosides salvage and systemic lupus erythematosus were the most significantly enriched for DEGs (up and down regulated). Blood vessel morphogenesis and cell cycle phase transition were the most significantly enriched term in biological processes, while extracellular matrix and chromosome, centromeric region were in cellular component and heparin binding and protein dimerization activity were in molecular function. Among the PPI networks and its module, target genes-miRNA regulatory network and target genes-TF regulatory network, hub genes were YWHAZ, FN1, GRK5, VCAM1, GATA6, TXNIP, HSPA1A, and F11R. Hub genes such as YWHAZ, STAT1, ICAM1, SH3BP5, CD83, FN1, TK1, HIST1H1C, CABLES1, and MCM3 were associated with poor overall survival, while hub genes such as STAT1, ICAM1, CD83, FN1, TK1, HIST1H1C, and MCM3 were highly expressed in stage 4. In conclusion, DEGs and hub genes diagnosed in this study may deepen our understanding of molecular mechanisms underlying the progression of ACC, and provide important targets for diagnosis and treatment of ACC.

Amino acid residues involved in the heparin-binding activity of murine IL-12 in the context of an antibody-cytokine fusion protein

An antibody-cytokine fusion protein, composed of the murine single-chain cytokine interleukin-12 (IL-12) genetically fused to a human IgG3 specific for the human tumor-associated antigen HER2/neu maintains antigen binding, cytokine bioactivity, and IL-12 heparin-binding activity. This latter property is responsible for the binding of the cytokine to glycosaminoglycans (GAGs) on the cell surface and the extracellular matrix and has been implicated in modulating IL-12 bioactivity. Previous studies indicate that the p40 subunit of human and murine IL-12 is responsible for the heparin-binding activity of this heterodimeric cytokine. In the present study we used bioinformatic analysis and site-directed mutagenesis to develop a version of the antibody-(IL-12) fusion protein without heparin-binding activity. This was accomplished by replacing the basic arginine (R) and lysine (K) residues in the cluster of amino acids 254–260 (RKKEKMK) of the murine IL-12 p40 subunit by the neutral non-polar amino acid alanine (A), generating an AAAEAMA mutant fusion protein. ELISA and flow cytometry demonstrated that the antibody fusion protein lacks heparin-binding activity but retains antigen binding. A T-cell proliferation assay showed IL-12 bioactivity in this construct. However, the IL-12 bioactivity is decreased compared to its non-mutated counterpart, which is consistent with an ancillary role of the heparin-binding site of IL-12 in modulating its activity. Thus, we have defined a cluster of amino acid residues with a crucial role in the heparin-binding activity of murine IL-12 in the context of an antibody-cytokine fusion protein.

Site-directed mutagenesis of cysteine to serine residues affects heparin binding and mitogenicity in fibroblast growth factor 4 produced in Escherichia coli

The binding activity to heparan sulphate is crucial for the mitogenic activity of fibroblast growth factor 4 (FGF4) in developing mammalian embryos. There are two conserved cysteine residues in FGF family proteins, Cys-84 and Cys-151 in mouse FGF4, and these constitute all of the cysteine residues in FGF4. However, the relationships among the heparin binding activity, growth promoting activity, and the two conserved cysteine residues in FGF4 are still unclear. Consequently, we generated in Escherichia coli three kinds of point-mutated mouse FGF4, namely C84S, C151S, and C84S;C151S, by converting the cysteine residues to serine residues. In heparin column chromatography, the heparin binding activities of these mutants were attenuated. In particular, the activity of the double-mutated C84S;C151S was weakened considerably. The growth promoting activities of these mutants correlated well with their heparin binding activities. We also demonstrated that an octapeptide, the Leu-76 to Tyr-83 region, which contained four basic amino acid residues and flanked Cys-84 in mouse FGF4, enhanced the heparin binding activity when fused to glutathione-S-transferase as a recombinant protein. Overall, our findings imply that the two conserved cysteine residues in FGF4 are both involved in the heparin binding activity and mitogenicity likely by affecting the configuration of heparin binding sites in FGF4.

A hepatitis B virus-derived human hepatic cell-specific heparin-binding peptide: identification and application to a drug delivery system.

Viruses are naturally evolved nanocarriers that can evade host immune systems, attach specifically to the surfaces of target cells, enter the cells through endocytosis, escape from endosomes efficiently, and then transfer their genomes to host cells. Hepatitis B virus (HBV) is a ∼42 nm enveloped DNA virus that can specifically infect human hepatic cells. To utilize the HBV-derived early infection machinery in synthetic nanocarriers, the human hepatic cell-binding site (i.e., the sodium taurocholate co-transporting polypeptide (NTCP)-binding site, with myristoylated pre-S1(2-47)) and the low pH-dependent fusogenic domain (pre-S1(9-24)) are indispensable for targeting and endosomal escape, respectively. However, cell-surface NTCP has recently been shown not to be involved in the initial attachment of HBV. In this study, we identified a novel heparin-binding site (pre-S1(30-42)) in the N-terminal half of the pre-S1 region, which presumably interacts with cell-surface heparan sulfate proteoglycan (HSPG) and plays a pivotal role in the initial attachment of HBV to human hepatic cells. The evolutionarily conserved amino acid residues Asp-31, Trp-32, and Asp-33 are indispensable for the heparin-binding activity. Liposomes (LPs) displaying the peptide were endocytosed by human hepatic cells in a cell-surface heparin-dependent manner and delivered doxorubicin to human hepatic cells more efficiently than myristoylated pre-S1(2-47)-displaying LPs. These results demonstrated that the pre-S1(30-42) peptide is the most promising HBV-derived targeting peptide for synthetic nanocarriers, and that this peptide exhibits high specificity for human hepatic cells and efficiently induces endocytosis.

Lectin ZG16p inhibits proliferation of human colorectal cancer cells via its carbohydrate-binding sites.

Zymogen granule protein 16 (ZG16p) is a soluble lectin that binds to both mannose and heparin/heparan sulfate. It is highly expressed in the human digestive tract and is secreted into the mucus. In this study, we investigated the effect of ZG16p on the proliferation of human colorectal cancer cells. Overexpression of ZG16p in Caco-2 cells decreased cell growth. Recombinant ZG16p markedly inhibited proliferation of Caco-2, LS174T, HCT116 and HCT15 cells. Caco-2 cell growth was not inhibited by two mutated ZG16p proteins, D151A and M5 (K36A, R37A, R53A, R55A and R79A) lacking mannose- and heparin-binding activities, respectively. Immunofluorescent cell staining revealed that ZG16p-D151A maintained its binding to the Caco-2 cell surface, whereas ZG16p-M5 failed to bind to the cells. These results suggest that ZG16p interacts with the cell surface via basic amino acids substituted in ZG16p-M5 and inhibits Caco-2 cell proliferation via Asp151. In addition, growth of patient-derived colorectal tumor organoids in a 3D intestinal stem cell system was suppressed by ZG16p but not by ZG16p-M5. Taken together, our findings indicate that ZG16p inhibits the growth of colorectal cancer cells via its carbohydrate-binding sites in vitro and ex vivo. In this study, a novel pathway in cancer cell growth regulation through cell surface carbohydrate chains is suggested.

Effects of oolonghomobisflavan A on oxidation of low-density lipoprotein.

Oxidation of low-density lipoprotein (LDL) by reactive oxygen species (ROS) and reactive nitrogen species (RNS) has been suggested to be involved in the onset of atherosclerosis. Oolong tea contains unique polyphenols including oolonghomobisflavan A (OFA). In this study, the effects of OFA on LDL oxidation by ROS and RNS were investigated in vitro. OFA suppressed formation of cholesterol ester hydroperoxides in LDL oxidized by peroxyl radical and peroxynitrite, and formation of thiobarbituric acid reactive substances in LDL oxidized by Cu2+. In addition, OFA inhibited fragmentation, carbonylation, and nitration of apolipoprotein B-100 (apo B-100) in the oxidized LDL, in which heparin-binding activity of apo B-100 was protected by OFA. Our results suggest that OFA exhibits antioxidant activity against both lipid peroxidation and oxidative modification of apo B-100 in LDL oxidized by ROS and RNS. Polyphenols in oolong tea may prevent atherosclerosis by reducing oxidative stress.

Dual-function synthetic peptide derived from BMP4 for highly efficient tumor targeting and antiangiogenesis.

Angiogenesis plays a critical role in the growth and metastasis of cancer, and growth factors released from cancer promote blood-vessel formation in the tumor microenvironment. The angiogenesis is accelerated via interactions of growth factors with the high-affinity receptors on cancer cells. In particular, heparan sulfate proteoglycans (HSPGs) on the surface of cancer cells have been shown to be important in many aspects of determining a tumor’s phenotype and development. Specifically, the regulation of the interactions between HSPGs and growth factors results in changes in tumor progression. A peptide with heparin-binding (HBP) activity has been developed and synthesized to inhibit tumor growth via the prevention of angiogenesis. We hypothesized that HBP could inhibit the interaction of growth factors and HSPGs on the surface of cancer cells, decrease paracrine signaling in endothelial cells (ECs), and finally decrease angiogenesis in the tumor microenvironment. In this study, we found that HBP had antiangiogenic effects in vitro and in vivo. The conditioned media obtained from a breast cancer cell line treated with HBP were used to culture human umbilical vein ECs (HUVECs) to evaluate the antiangiogenic effect of HBP on ECs. HBP effectively inhibited the migration, invasion, and tube formation of HUVECs in vitro. In addition, the expressions of angiogenesis-mediating factors, including ERK, FAK, and Akt, were considerably decreased. HBP also decreased the levels of invasive factors, including MMP2 and MMP9, secreted by the HUVECs. We demonstrated significant suppression of tumor growth in a breast cancer xenograft model and enhanced distribution of HBP at the site of tumors. Taken together, our results show that HBP has antiangiogenic effects on ECs, and suggest that it may serve as a potential antitumor agent through control of the tumor microenvironment.

The Vaccinia Virus H3 Envelope Protein, a Major Target of Neutralizing Antibodies, Exhibits a Glycosyltransferase Fold and Binds UDP-Glucose.

The highly conserved H3 poxvirus protein is a major target of the human antibody response against poxviruses and is likely a key contributor to protection against infection. Here, we present the crystal structure of H3 from vaccinia virus at a 1.9-Å resolution. H3 looks like a glycosyltransferase, a family of enzymes that transfer carbohydrate molecules to a variety of acceptor substrates. Like glycosyltransferases, H3 binds UDP-glucose, as shown by saturation transfer difference (STD) nuclear magnetic resonance (NMR) spectroscopy, and this binding requires Mg(2+) Mutation of the glycosyltransferase-like metal ion binding motif in H3 greatly diminished its binding to UDP-glucose. We found by flow cytometry that H3 binds to the surface of human cells but does not bind well to cells that are deficient in surface glycosaminoglycans. STD NMR experiments using a heparin sulfate decasaccharide confirmed that H3 binds heparin sulfate. We propose that a surface of H3 with an excess positive charge may be the binding site for heparin. Heparin binding and glycosyltransferase activity may be involved in the function of H3 in the poxvirus life cycle. Poxviruses are under intense research because of bioterrorism concerns, zoonotic infections, and the side effects of existing smallpox vaccines. The smallpox vaccine using vaccinia virus has been highly successful, but it is still unclear why the vaccine is so effective. Studying the antigens that the immune system recognizes may allow a better understanding of how the vaccine elicits immunity and how improved vaccines can be developed. Poxvirus protein H3 is a major target of the immune system. The H3 crystal structure shows that it has a glycosyltransferase protein fold. We demonstrate that H3 binds the sugar nucleotide UDP-glucose, as do glycosyltransferases. Our experiments also reveal that H3 binds cell surface molecules that are involved in the attachment of poxviruses to cells. These structural and functional studies of H3 will help in designing better vaccines and therapeutics.

Mapping the heparin-binding site of the osteoinductive protein NELL1 by site-directed mutagenesis

Neural epidermal growth factor-like (NEL)-like 1 (NELL1) is a secretory osteogenic protein comprising an N-terminal thrombospondin-1-like (TSPN) domain, four von Willebrand factor type C domains, and six epidermal growth factor-like repeats. NELL1 shows heparin-binding activity; however, the biological significance remains to be explored. In this report, we demonstrate that NELL1 binds to cell surface proteoglycans through its TSPN domain. Major heparin-binding sites were identified on the three-dimensional structural model of the TSPN domain of NELL1. Mutant analysis of the heparin-binding sites indicated that the heparin-binding activity of the TSPN domain is involved in interaction of NELL1 with cell surface proteoglycans.

Vascular endothelial growth factor from Trimeresurus jerdonii venom specifically binds to VEGFR-2.

Vascular endothelial growth factors (VEGFs) play important roles in angiogenesis. In this study, a vascular endothelial growth factor named TjsvVEGF was purified from the venom of Trimeresurus jerdonii by gel filtration, affinity, ion-exchange and high-performance liquid chromatography. TjsvVEGF was a homodimer with an apparent molecular mass of 29kDa. The cDNA encoding TjsvVEGF was obtained by PCR. The open reading frame of the cloned TjsvVEGF was composed of 432 bp coding for a signal peptide of 24 amino acid residues and a mature protein of 119 amino acid residues. Compared with other snake venom VEGFs, the nucleotide and deduced protein sequences of the cloned TjsvVEGF were conserved. TjsvVEGF showed low heparin binding activity and strong capillary permeability increasing activity. The KD of TjsvVEGF to VEFGR-2 is 413 pM. However, the binding of TjsvVEGF to VEGFR-1 is too weak to detect. Though TjsvVEGF had high sequence identities (about 90%) with Crotalinae VEGFs, the receptor preference of TjsvVEGF was similar to Viperinae VEGFs which had lower sequence identities (about 60%) with it. TjsvVEGF might serve as a useful tool for the study of structure-function relationships of VEGFs and their receptors.

Сіальованість глікопротеїнів і рівень експресії нейрамінідази NEU1 та сіалілтрансферази ST6GAL1 у лімфоцитах хворих на еритремію

We studied the levels of lymphocyte surface-associated sialylglycans and the expression of neuraminidase NEU1 and sialyltransferase ST6GAL1 genes in lymphocytes in erythremia patients and healthy donors as well as the levels of sialic acids in plasma and sialylation of alpha-acid glycoprotein and fibronectin. Moreover, we also investigated the type of sialic acids binding with its glycans using sialospecific lectins MAA and SNA. fibronectin protein in lymphocytes and its cell surface in erythremia disease as compared to healthy donors. It was shown that the levels of free sialic acids and neuraminidase activity in plasma are increased in erythremia disease as compared to healthy donors; however, MAA-II-binding activity of tested glycoproteins is decreased, fibronectin-1 mRNA expression in lymphocytes is increased in patients with erythremia. The decreasing of plasma fibronectin concentration and its heparin-binding activity as well as increasing of lymphocyte content with surface-associated and intracellular fibronectin were revealed in erythremia disease in comparison with healthy donors. Positive correlation between plasma fibronectin level and its heparin-binding activity and negative correlation between plasma fibronectin level and quantity of lymphocytes which express fibronectin inside the cell and on cell surface was detected. Enhanced levels of α2,3- and α2,6-linked residues of glycocojugates were detected on lymphocyte cell surface in erythremia disease using sialospecific lectins and flow cytometer as well as fluorescent confocal microscope. The level of NEU1 and ST6GAL1 mRNA expressions is significantly increased in lymphocytes in erythremia disease. Results of this study are clarified the mechanisms of disturbed in erythremia disease glycobiological processes and may therefore present new approaches for therapeutic opportunities.

Antimicrobial Effects of Helix D-derived Peptides of Human Antithrombin III

Antithrombin III (ATIII) is a key antiproteinase involved in blood coagulation. Previous investigations have shown that ATIII is degraded by Staphylococcus aureus V8 protease, leading to release of heparin binding fragments derived from its D helix. As heparin binding and antimicrobial activity of peptides frequently overlap, we here set out to explore possible antibacterial effects of intact and degraded ATIII. In contrast to intact ATIII, the results showed that extensive degradation of the molecule yielded fragments with antimicrobial activity. Correspondingly, the heparin-binding, helix D-derived, peptide FFFAKLNCRLYRKANKSSKLV (FFF21) of human ATIII, was found to be antimicrobial against particularly the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa. Fluorescence microscopy and electron microscopy studies demonstrated that FFF21 binds to and permeabilizes bacterial membranes. Analogously, FFF21 was found to induce membrane leakage of model anionic liposomes. In vivo, FFF21 significantly reduced P. aeruginosa infection in mice. Additionally, FFF21 displayed anti-endotoxic effects in vitro. Taken together, our results suggest novel roles for ATIII-derived peptide fragments in host defense.

Follistatin: A Novel Therapeutic for the Improvement of Muscle Regeneration

Follistatin (FST) is a member of the tissue growth factor β family and is a secreted glycoprotein that antagonizes many members of the family, including activin A, growth differentiation factor 11, and myostatin. The objective of this study was to explore the use of an engineered follistatin therapeutic created by fusing FST315 lacking heparin binding activity to the N terminus of a murine IgG1 Fc (FST315-ΔHBS-Fc) as a systemic therapeutic agent in models of muscle injury. Systemic administration of this molecule was found to increase body weight and lean muscle mass after weekly administration in normal mice. Subsequently, we tested this agent in several models of muscle injury, which were chosen based on their severity of damage and their ability to reflect clinical settings. FST315-ΔHBS-Fc treatment proved to be a potent inducer of muscle remodeling and regeneration. FST315-ΔHBS-Fc induced improvements in muscle repair after injury/atrophy by modulating the early inflammatory phase allowing for increased macrophage density, and Pax7-positive cells leading to an accelerated restoration of myofibers and muscle function. Collectively, these data demonstrate the benefits of a therapeutically viable form of FST that can be leveraged as an alternate means of ameliorating muscle regeneration.

Chondroitin Sulfate is the Primary Receptor for a Peptide-Modified AAV That Targets Brain Vascular Endothelium In Vivo

Recently, we described a peptide-modified AAV2 vector (AAV-GMN) containing a capsid-displayed peptide that directs in vivo brain vascular targeting and transduction when delivered intravenously. In this study, we sought to identify the receptor that mediates transduction by AAV-GMN. We found that AAV-GMN, but not AAV2, readily transduces the murine brain endothelial cell line bEnd.3, a result that mirrors previously observed in vivo transduction profiles of brain vasculature. Studies in vitro revealed that the glycosaminoglycan, chondroitin sulfate C, acts as the primary receptor for AAV-GMN. Unlike AAV2, chondroitin sulfate expression is required for cell transduction by AAV-GMN, and soluble chondroitin sulfate C can robustly inhibit AAV-GMN transduction of brain endothelial cells. Interestingly, AAV-GMN retains heparin-binding properties, though in contrast to AAV2, it poorly transduces cells that express heparan sulfate but not chondroitin sulfate, indicating that the peptide insertion negatively impacts heparan-mediated transduction. Lastly, when delivered directly, this modified virus can transduce multiple brain regions, indicating that the potential of AAV-GMN as a therapeutic gene delivery vector for central nervous system disorders is not restricted to brain vascular endothelium.

Purification of Vitronectin

This unit describes the purification of extracellular vitronectin from plasma or serum by using heparin-affinity chromatography. First, the plasma is depleted of fibronectin plus other heparin- and Sepharose-binding proteins and treated with urea to activate the heparin-binding activity of vitronectin, which is subsequently bound to a heparin affinity column and eluted. The resulting vitronectin should be ∼ 98% pure.

The neuroprotective activity of the amyloid precursor protein against traumatic brain injury is mediated via the heparin binding site in residues 96‐110

We have previously shown that following traumatic brain injury (TBI) the presence of the amyloid precursor protein (APP) may be neuroprotective. APP knockout mice have increased neuronal death and worse cognitive and motor outcomes following TBI, which is rescued by treatment with exogenous sAPPα (the secreted ectodomain of APP generated by α-secretase cleavage). Two neuroprotective regions were identified in sAPPα, the N and C-terminal domains D1 and D6a/E2 respectively. As both D1 and D6a/E2 contain heparin binding activity it was hypothesized that this is responsible for the neuroprotective activity. In this study, we focused on the heparin binding site, encompassed by residues 96-110 in D1, which has previously been shown to have neurotrophic properties. We found that treatment with APP96-110 rescued motor and cognitive deficits in APP-/- mice following focal TBI. APP96-110 also provided neuroprotection in Sprague-Dawley rats following diffuse TBI. Treatment with APP96-110 significantly improved functional outcome as well as preserve histological cellular morphology in APP-/- mice following focal controlled cortical impact injury. Furthermore, following administration of APP96-110 in rats after diffuse impact acceleration TBI, motor and cognitive outcomes were significantly improved and axonal injury reduced. These data define the heparin binding site in the D1 domain of sAPPα, represented by the sequence APP96-110, as the neuroprotective site to confer neuroprotection following TBI. The product of α-secretase cleavage of the amyloid precursor protein, sAPPα is neuroprotective following traumatic brain injury (TBI). Of interest was whether this neuroprotective activity could be further delineated to a heparin binding region within sAPPα, corresponding to the region APP96-110 (see diagram demonstrating the domain structure of sAPPα). Indeed treatment with APP96-110 improved functional outcome following TBI, an effect that was not seen with a mutated version of the peptide that had reduced heparin binding affinity.