Heparan sulfate-dependent phase separation of CCL5 and its chemotactic activity.

Secreted chemokines form concentration gradients in target tissues to control migratory directions and patterns of immune cells in response to inflammatory stimulation; however, how the gradients are formed is much debated. Heparan sulfate (HS) binds to chemokines and modulates their activities. In this study, we investigated the roles of HS in the gradient formation and chemoattractant activity of CCL5 that is known to bind to HS. CCL5 and heparin underwent liquid-liquid phase separation and formed gradient, which was confirmed using CCL5 immobilized on heparin-beads. The biological implication of HS in CCL5 gradient formation was established in CHO-K1 (wild-type) and CHO-677 (lacking HS) cells by Transwell assay. The effect of HS on CCL5 chemoattractant activity was further proved by Transwell assay of human peripheral blood cells. Finally, peritoneal injection of the chemokines into mice showed reduced recruitment of inflammatory cells either by mutant CCL5 (lacking heparin-binding sequence) or by addition of heparin to wild-type CCL5. Our experimental data propose that co-phase separation of CCL5 with HS establishes a specific chemokine concentration gradient to trigger directional cell migration. The results warrant further investigation on other heparin-binding chemokines and allows for a more elaborate insight into disease process and new treatment strategies.

Identification of heparin-binding amino acid residues in antibody HS4C3 with the potential to design antibodies against heparan sulfate domains.

Heparan sulfate (HS) is a linear polysaccharide with high structural and functional diversity. Detection and localization of HS in tissues can be performed using single chain variable fragment (scFv) antibodies. Although several anti-HS antibodies recognizing different sulfation motifs have been identified, little is known about their interaction with HS. In this study the interaction between the scFv antibody HS4C3 and heparin was investigated. Heparin-binding lysine and arginine residues were identified using a protect and label methodology. Site-directed mutagenesis was applied to further identify critical heparin-binding lysine/arginine residues using immunohistochemical and biochemical assays. In addition, computational docking of a heparin tetrasaccharide towards a 3-D homology model of HS4C3 was applied to identify potential heparin-binding sites. Of the 12 lysine and 15 arginine residues within the HS4C3 antibody, 6 and 9, respectively, were identified as heparin-binding. Most of these residues are located within one of the complementarity determining regions (CDR) or in their proximity. All basic amino acid residues in the CDR3 region of the heavy chain were involved in binding. Computational docking showed a heparin tetrasaccharide close to these regions. Mutagenesis of heparin-binding residues reduced or altered reactivity towards HS and heparin. Identification of heparin-binding arginine and lysine residues in HS4C3 allows for better understanding of the interaction with HS and creates a framework to rationally design antibodies targeting specific HS motifs.

Molecular Mechanisms of the Impaired Heparin Pentasaccharide Interactions in 10 Antithrombin Heparin Binding Site Mutants Revealed by Enhanced Sampling Molecular Dynamics.

Antithrombin (AT) is a critical regulator of the coagulation cascade by inhibiting multiple coagulation factors including thrombin and FXa. Binding of heparinoids to this serpin enhances the inhibition considerably. Mutations located in the heparin binding site of AT result in thrombophilia in affected individuals. Our aim was to study 10 antithrombin mutations known to affect their heparin binding in a heparin pentasaccharide bound state using two molecular dynamics (MD) based methods providing enhanced sampling, GaMD and LiGaMD2. The latter provides an additional boost to the ligand and the most important binding site residues. From our GaMD simulations we were able to identify four variants (three affecting amino acid Arg47 and one affecting Lys114) that have a particularly large effect on binding. The additional acceleration provided by LiGaMD2 allowed us to study the consequences of several other mutants including those affecting Arg13 and Arg129. We were able to identify several conformational types by cluster analysis. Analysis of the simulation trajectories revealed the causes of the impaired pentasaccharide binding including pentasaccharide subunit conformational changes and altered allosteric pathways in the AT protein. Our results provide insights into the effects of AT mutations interfering with heparin binding at an atomic level and can facilitate the design or interpretation of in vitro experiments.

Chemokine Oligomers and the Impact of Fondaparinux Binding.

Heparin, a widely used clinical anticoagulant, is generally well-tolerated; however, approximately 1% of patients develop heparin-induced thrombocytopenia (HIT), a serious side effect. While efforts to understand the role of chemokines in HIT development are ongoing, certain aspects remain less studied, such as the stabilization of chemokine oligomers by heparin. Here, we conducted a combined ion mobility-native mass spectrometry study to investigate the stability of chemokine oligomers and their complexes with fondaparinux, a synthetic heparin analog. Collision-induced dissociation and unfolding experiments provided clarity on the specificity and relevance of chemokine oligomers and their fondaparinux complexes with varying stoichiometries, as well as the stabilizing effects of fondaparinux binding.

Differential Solvent DEEP-STD NMR and MD Simulations Enable the Determinants of the Molecular Recognition of Heparin Oligosaccharides by Antithrombin to Be Disentangled.

The interaction of heparin with antithrombin (AT) involves a specific sequence corresponding to the pentasaccharide GlcNAc/NS6S-GlcA-GlcNS3S6S-IdoA2S-GlcNS6S (AGA*IA). Recent studies have revealed that two AGA*IA-containing hexasaccharides, which differ in the sulfation degree of the iduronic acid unit, exhibit similar binding to AT, albeit with different affinities. However, the lack of experimental data concerning the molecular contacts between these ligands and the amino acids within the protein-binding site prevents a detailed description of the complexes. Differential epitope mapping (DEEP)-STD NMR, in combination with MD simulations, enables the experimental observation and comparison of two heparin pentasaccharides interacting with AT, revealing slightly different bound orientations and distinct affinities of both glycans for AT. We demonstrate the effectiveness of the differential solvent DEEP-STD NMR approach in determining the presence of polar residues in the recognition sites of glycosaminoglycan-binding proteins.

Biosynthetic production of anticoagulant heparin polysaccharides through metabolic and sulfotransferases engineering strategies.

Heparin is an important anticoagulant drug, and microbial heparin biosynthesis is a potential alternative to animal-derived heparin production. However, effectively using heparin synthesis enzymes faces challenges, especially with microbial recombinant expression of active heparan sulfate N-deacetylase/N-sulfotransferase. Here, we introduce the monosaccharide N-trifluoroacetylglucosamine into Escherichia coli K5 to facilitate sulfation modification. The Protein Repair One-Stop Service-Focused Rational Iterative Site-specific Mutagenesis (PROSS-FRISM) platform is used to enhance sulfotransferase efficiency, resulting in the engineered NST-M8 enzyme with significantly improved stability (11.32-fold) and activity (2.53-fold) compared to the wild-type N-sulfotransferase. This approach can be applied to engineering various sulfotransferases. The multienzyme cascade reaction enables the production of active heparin from bioengineered heparosan, demonstrating anti-FXa (246.09 IU/mg) and anti-FIIa (48.62 IU/mg) activities. This study offers insights into overcoming challenges in heparin synthesis and modification, paving the way for the future development of animal-free heparins using a cellular system-based semisynthetic strategy.

Exploring single-molecule interactions: heparin and FGF-1 proteins through solid-state nanopores.

Detection and characterization of protein-protein interactions are essential for many cellular processes, such as cell growth, tissue repair, drug delivery, and other physiological functions. In our research, we have utilized emerging solid-state nanopore sensing technology, which is highly sensitive to better understand heparin and fibroblast growth factor 1 (FGF-1) protein interactions at a single-molecule level without any modifications. Understanding the structure and behavior of heparin-FGF-1 complexes at the single-molecule level is very important. An abnormality in their formation can lead to life-threatening conditions like tumor growth, fibrosis, and neurological disorders. Using a controlled dielectric breakdown pore fabrication approach, we have characterized individual heparin and FGF-1 (one of the 22 known FGFs in humans) proteins through the fabrication of 17 ± 1 nm nanopores. Compared to heparin, the positively charged heparin-binding domains of some FGF-1 proteins translocationally react with the pore walls, giving rise to a distinguishable second peak with higher current blockade. Additionally, we have confirmed that the dynamic FGF-1 is stabilized upon binding with heparin-FGF-1 at the single-molecule level. The larger current blockades from the complexes relative to individual heparin and the FGF-1 recorded during the translocation ensure the binding of heparin-FGF-1 proteins, forming binding complexes with higher excluded volumes. Taken together, we demonstrate that solid-state nanopores can be employed to investigate the properties of individual proteins and their complex interactions, potentially paving the way for innovative medical therapies and advancements.

Marine sulfated glycans inhibit the interaction of heparin with S-protein of SARS-CoV-2 Omicron XBB variant.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a worldwide COVID-19 pandemic, leading to 6.8 million deaths. Numerous variants have emerged since its outbreak, resulting in its significantly enhanced ability to spread among humans. As with many other viruses, SARS­CoV­2 utilizes heparan sulfate (HS) glycosaminoglycan (GAG) on the surface of host cells to facilitate viral attachment and initiate cellular entry through the ACE2 receptor. Therefore, interfering with virion-HS interactions represents a promising target to develop broad-spectrum antiviral therapeutics. Sulfated glycans derived from marine organisms have been proven to be exceptional reservoirs of naturally existing HS mimetics, which exhibit remarkable therapeutic properties encompassing antiviral/microbial, antitumor, anticoagulant, and anti-inflammatory activities. In the current study, the interactions between the receptor-binding domain (RBD) of S-protein of SARS-CoV-2 (both WT and XBB.1.5 variants) and heparin were applied to assess the inhibitory activity of 10 marine-sourced glycans including three sulfated fucans, three fucosylated chondroitin sulfates and two fucoidans derived from sea cucumbers, sea urchin and seaweed Saccharina japonica, respectively. The inhibitory activity of these marine derived sulfated glycans on the interactions between RBD of S-protein and heparin was evaluated using Surface Plasmon Resonance (SPR). The RBDs of S-proteins from both Omicrion XBB.1.5 and wild-type (WT) were found to bind to heparin, which is a highly sulfated form of HS. All the tested marine-sourced sulfated glycans exhibited strong inhibition of WT and XBB.1.5 S-protein binding to heparin. We believe the study on the molecular interactions between S-proteins and host cell glycosaminoglycans provides valuable insight for the development of marine-sourced, glycan-based inhibitors as potential anti-SARS-CoV-2 agents.

Synthesis of bioengineered heparin chemically and biologically similar to porcine-derived products and convertible to low MW heparin.

Heparins have been invaluable therapeutic anticoagulant polysaccharides for over a century, whether used as unfractionated heparin or as low molecular weight heparin (LMWH) derivatives. However, heparin production by extraction from animal tissues presents multiple challenges, including the risk of adulteration, contamination, prion and viral impurities, limited supply, insecure supply chain, and significant batch-to-batch variability. The use of animal-derived heparin also raises ethical and religious concerns, as well as carries the risk of transmitting zoonotic diseases. Chemoenzymatic synthesis of animal-free heparin products would offer several advantages, including reliable and scalable production processes, improved purity and consistency, and the ability to produce heparin polysaccharides with molecular weight, structural, and functional properties equivalent to those of the United States Pharmacopeia (USP) heparin, currently only sourced from porcine intestinal mucosa. We report a scalable process for the production of bioengineered heparin that is biologically and compositionally similar to USP heparin. This process relies on enzymes from the heparin biosynthetic pathway, immobilized on an inert support and requires a tailored N-sulfoheparosan with N-sulfo levels similar to those of porcine heparins. We also report the conversion of our bioengineered heparin into a LMWH that is biologically and compositionally similar to USP enoxaparin. Ultimately, we demonstrate major advances to a process to provide a potential clinical and sustainable alternative to porcine-derived heparin products.

A type I interferon regulatory network for human plasmacytoid dendritic cells based on heparin, membrane-bound and soluble BDCA-2.

Plasmacytoid dendritic cells (pDCs) produce type I interferons (IFNs) after sensing viral/bacterial RNA or DNA by toll-like receptor (TLR) 7 or TLR9, respectively. However, aberrant pDCs activation can cause adverse effects on the host and contributes to the pathogenesis of type I IFN-related autoimmune diseases. Here, we show that heparin interacts with the human pDCs-specific blood dendritic cell antigen 2 (BDCA-2) but not with related lectins such as DCIR or dectin-2. Importantly, BDCA-2-heparin interaction depends on heparin sulfation and receptor glycosylation and results in inhibition of TLR9-driven type I IFN production in primary human pDCs and the pDC-like cell line CAL-1. This inhibition is mediated by unfractionated and low-molecular-weight heparin, as well as endogenous heparin from plasma, suggesting that the local blood environment controls the production of IFN-α in pDCs. Additionally, we identified an activation-dependent soluble form of BDCA-2 (solBDCA-2) in human plasma that functions as heparin antagonist and thereby increases TLR9-driven IFN-α production in pDCs. Of importance, solBDCA-2 levels in the serum were increased in patients with scrub typhus (an acute infectious disease caused by Orientia tsutsugamushi) compared to healthy control subjects and correlated with anti-dsDNA antibodies titers. In contrast, solBDCA-2 levels in plasma from patients with bullous pemphigoid or psoriasis were reduced. In summary, this work identifies a regulatory network consisting of heparin, membrane-bound and solBDCA-2 modulating TLR9-driven IFN-α production in pDCs. This insight into pDCs function and regulation may have implications for the treatment of pDCs-related autoimmune diseases.

Clinical and Molecular Characterization of Nine Novel Antithrombin Mutations.

Antithrombin (AT) is the major plasma inhibitor of thrombin (FIIa) and activated factor X (FXa), and antithrombin deficiency (ATD) is one of the most severe thrombophilic disorders. In this study, we identified nine novel AT mutations and investigated their genotype-phenotype correlations. Clinical and laboratory data from patients were collected, and the nine mutant AT proteins (p.Arg14Lys, p.Cys32Tyr, p.Arg78Gly, p.Met121Arg, p.Leu245Pro, p.Leu270Argfs*14, p.Asn450Ile, p.Gly456delins_Ala_Thr and p.Pro461Thr) were expressed in HEK293 cells; then, Western blotting, N-Glycosidase F digestion, and ELISA were used to detect wild-type and mutant AT. RT-qPCR was performed to determine the expression of AT mRNA from the transfected cells. Functional studies (AT activity in the presence and in the absence of heparin and heparin-binding studies with the surface plasmon resonance method) were carried out. Mutations were also investigated by in silico methods. Type I ATD caused by altered protein synthesis (p.Cys32Tyr, p.Leu270Argfs*14, p.Asn450Ile) or secretion disorder (p.Met121Arg, p.Leu245Pro, p.Gly456delins_Ala_Thr) was proved in six mutants, while type II heparin-binding-site ATD (p.Arg78Gly) and pleiotropic-effect ATD (p.Pro461Thr) were suggested in two mutants. Finally, the pathogenic role of p.Arg14Lys was equivocal. We provided evidence to understand the pathogenic nature of novel SERPINC1 mutations through in vitro expression studies.

Advancements in heparosan production through metabolic engineering and improved fermentation.

Heparin is one of the most widely used natural drugs, and has been the preferred anticoagulant and antithrombotic agent in the clinical setting for nearly a century. Heparin also shows increasing therapeutic potential for treating inflammation, cancer, and microbial and viral diseases, including COVID-19. With advancements in synthetic biology, heparin production through microbial engineering of heparosan offers a cost-effective and scalable alternative to traditional extraction from animal tissues. Heparosan serves as the starting carbon backbone for the chemoenzymatic synthesis of bioengineered heparin, possessing a chain length that is critically important for the production of heparin-based therapeutics with specific molecular weight (MW) distributions. Recent advancements in metabolic engineering of microbial cell factories have resulted in high-yield heparosan production. This review systematically analyzes the key modules involved in microbial heparosan biosynthesis and the latest metabolic engineering strategies for enhancing production, regulating MW, and optimizing the fermentation scale-up of heparosan. It also discusses future studies, remaining challenges, and prospects in the field.

Natural carboxyterminal truncation of human CXCL10 attenuates glycosaminoglycan binding, CXCR3A signaling and lymphocyte chemotaxis, while retaining angiostatic activity.

BACKGROUND: Interferon-γ-inducible protein of 10 kDa (IP-10/CXCL10) is a dual-function CXC chemokine that coordinates chemotaxis of activated T cells and natural killer (NK) cells via interaction with its G protein-coupled receptor (GPCR), CXC chemokine receptor 3 (CXCR3). As a consequence of natural posttranslational modifications, human CXCL10 exhibits a high degree of structural and functional heterogeneity. However, the biological effect of natural posttranslational processing of CXCL10 at the carboxy (C)-terminus has remained partially elusive. We studied CXCL10(1-73), lacking the four endmost C-terminal amino acids, which was previously identified in supernatant of cultured human fibroblasts and keratinocytes. METHODS: Relative levels of CXCL10(1-73) and intact CXCL10(1-77) were determined in synovial fluids of patients with rheumatoid arthritis (RA) through tandem mass spectrometry. The production of CXCL10(1-73) was optimized through Fmoc-based solid phase peptide synthesis (SPPS) and a strategy to efficiently generate human CXCL10 proteoforms was introduced. CXCL10(1-73) was compared to intact CXCL10(1-77) using surface plasmon resonance for glycosaminoglycan (GAG) binding affinity, assays for cell migration, second messenger signaling downstream of CXCR3, and flow cytometry of CHO cells and primary human T lymphocytes and endothelial cells. Leukocyte recruitment in vivo upon intraperitoneal injection of CXCL10(1-73) was also evaluated. RESULTS: Natural CXCL10(1-73) was more abundantly present compared to intact CXCL10(1-77) in synovial fluids of patients with RA. CXCL10(1-73) had diminished affinity for GAG including heparin, heparan sulfate and chondroitin sulfate A. Moreover, CXCL10(1-73) exhibited an attenuated capacity to induce CXCR3A-mediated signaling, as evidenced in calcium mobilization assays and through quantification of phosphorylated extracellular signal-regulated kinase-1/2 (ERK1/2) and protein kinase B/Akt. Furthermore, CXCL10(1-73) incited significantly less primary human T lymphocyte chemotaxis in vitro and peritoneal ingress of CXCR3+ T lymphocytes in mice. In contrast, loss of the four endmost C-terminal residues did not affect the inhibitory properties of CXCL10 on migration, proliferation, wound closure, phosphorylation of ERK1/2, and sprouting of human microvascular endothelial cells. CONCLUSION: Our study shows that the C-terminal residues Lys74-Pro77 of CXCL10 are important for GAG binding, signaling through CXCR3A, T lymphocyte chemotaxis, but dispensable for angiostasis.

Synthesis and Detection of BODIPY-, Biotin-, and 19 F- Labeled Single-Entity Dendritic Heparan Sulfate Mimetics.

Heparin and heparan sulfate (HS) are naturally occurring mammalian glycosaminoglycans, and their synthetic and semi-synthetic mimetics have attracted significant interest as potential therapeutics. However, understanding the mechanism of action by which HS, heparin, and HS mimetics have a biological effect is difficult due to their highly charged nature, broad protein interactomes, and variable structures. To address this, a library of novel single-entity dendritic mimetics conjugated to BODIPY, Fluorine-19 (19 F), and biotin was synthesized for imaging and localization studies. The novel dendritic scaffold allowed for the conjugation of labeling moieties without reducing the number of sulfated capping groups, thereby better mimicking the multivalent nature of HS-protein interactions. The 19 F labeled mimetics were assessed in phantom studies and were detected at concentrations as low as 5 mM. Flow cytometric studies using a fluorescently labeled mimetic showed that the compound associated with immune cells from tumors more readily than splenic counterparts and was directed to endosomal-lysosomal compartments within immune cells and cancer cells. Furthermore, the fluorescently labeled mimetic entered the central nervous system and was detectable in brain-infiltrating immune cells 24 hours after treatment. Here, we report the enabling methodology for rapidly preparing various labeled HS mimetics and molecular probes with diverse potential therapeutic applications.

Scalable dual column cation exchange affinity chromatography based platform process for recombinant protein purification.

A novel tandem affinity tag is presented that enables the use of cation exchange resins for initial affinity purification, followed by an additional column step for enhanced purity and affinity tag self-removal. In this method, the highly charged heparin-binding tag binds strongly and selectively to either a strong or weak cation exchange resin based on electrostatic interactions, effectively acting as an initial affinity tag. Combining the heparin-binding tag (HB-tag) with the self-removing iCapTag™ provides a means for removing both tags in a subsequent self-cleaving step. The result is a convenient platform for the purification of diverse tagless proteins with a range of isoelectric points and molecular weights. In this work, we demonstrate a dual column process in which the tagged protein of interest is first captured from an E. coli cell lysate using a cation exchange column via a fused heparin-binding affinity tag. The partially purified protein is then diluted and loaded onto an iCapTag™ split-intein column, washed, and then incubated overnight to release the tagless target protein from the bound tag. Case studies are provided for enhanced green fluorescent protein (eGFP), beta galactosidase (ßgal), maltose binding protein (MBP) and beta lactamase (ßlac), where overall purity and host cell DNA clearance is provided. Overall, the proposed dual column process is shown to be a scalable platform technology capable of accessing both the high dynamic binding capacity of ion exchange resins and the high selectivity of affinity tags for the purification of recombinant proteins.

Lysine ε-aminolysis and incorporation of sulfhydryl groups into human brain tau 4R/1N and 306VQIVYK311 enhances the formation of beta structures and toxicity.

In the present study, we investigated the effects of N-homocysteine thiolactone (tHcy) modification on expressed and purified tau protein and the synthesized VQIVYK target peptide. The modified constructs were subjected to comprehensive validation using various methodologies, including mass spectrometry. Subsequently, in vivo, in vitro, and in silico characterizations were performed under both reducing and non-reducing conditions, as well as in the presence and absence of heparin as a cofactor. Our results unequivocally confirmed that under reducing conditions and in the presence of heparin, the modified constructs exhibited a greater propensity for aggregation. This enhanced aggregative behavior can be attributed to the disruption of lysine positive charges and the subsequent influence of hydrophobic and p-stacking intermolecular forces. Notably, the modified oligomeric species induced apoptosis in the SH-SY5Y cell line, and this effect was further exacerbated with longer incubation times and higher concentrations of the modifier. These observations suggest a potential mechanism involving reactive oxygen species (ROS). To gain a deeper understanding of the molecular mechanisms underlying the neurotoxic effects, further investigations are warranted. Elucidating these mechanisms will contribute to the development of more effective strategies to counteract aggregation and mitigate neurodegeneration.

Identification of heparin-binding proteins expressed on Trichosporon asahii cell surface.

Trichosporon asahii is a pathogenic yeast that cause trichosporonosis. T. asahii exhibits several colony morphologies, such as white (W)- or off-white (O)-type, which may affect virulence. In this study, we compared the expression pattern of heparin-binding proteins in various colony morphologies and identified heparin-binding protein in T. asahii. Surface plasmon resonance analysis revealed that cell surface molecules attached more strongly to heparin in W- than O-type cells. We purified and identified a heparin-binding protein strongly expressed in W-type cells using heparin-Sepharose beads, named it heparin-binding protein 1 (HepBP1), and expressed Flag-tagged HepBP1 in mammalian cells. The heparin-binding ability of Flag-tagged HepBP1 was confirmed by pulldown assay using heparin-Sepharose beads. Thus, HepBP1 is a heparin-binding protein on T. asahii cell surface. These results suggest that several T. asahii cell surface proteins interact with glycosaminoglycans; therefore, they could contribute to infection.

Heparinase III with High Activity and Stability: Heterologous Expression, Biochemical Characterization, and Application in Depolymerization of Heparin.

A novel heparinase III from Pedobacter schmidteae (PsHep-III) with high activity and good stability was successfully cloned, expressed, and characterized. PsHep-III displayed the highest specific activity ever reported of 192.8 U mg-1 using heparin as the substrate. It was stable at 25 °C with a half-life of 323 h in an aqueous solution. PsHep-III was employed for the depolymerization of heparin, and the enzymatic hydrolyzed products were analyzed with gel permeation chromatography and high-performance liquid chromatography. PsHep-III can break glycosidic bonds in heparin like →4]GlcNAc/GlcNAc6S/GlcNS/GlcNS6S/GlcN/GlcN6S(1 → 4)ΔUA/ΔUA2S[1 → and efficiently digest heparin into seven disaccharides including N-acetylated, N-sulfated, and N-unsubstituted modification, with molecular masses of 503, 605, 563, 563, 665, 360, and 563 Da, respectively. These results indicated that PsHep-III with broad substrate specificity could be combined with heparinase I to overcome the low selectivity at the N-acetylated modification binding sites of heparinase I. This work will contribute to the application of PsHep-III for characterizing heparin and producing low-molecular-weight heparin effectively.

Heparin Oligosaccharides as Vasoactive Intestinal Peptide Inhibitors via their Binding Process Characterization.

BACKGROUND: It has been proven that vasoactive intestinal peptide (VIP) was involved in the pathogenesis of prostate cancer. Cardin et al. found that by an alanine scan, the heparin- binding site on VIP was exactly the same sequence in VIP and its receptor. Therefore, heparin could competitively block the binding of VIP and its receptor. However, the structure-activity relationship between heparin and VIP has not been reported, especially in terms of the sequence and sulfation patterns of heparin oligosaccharides upon binding to VIP. OBJECTIVE: A variety of experiments were designed to study the binding process and structure-activity relationship between heparin oligosaccharides and VIP. METHODS: Heparin was enzymatically digested and purified to produce heparin oligosaccharides, and the structures were characterized by NMR. The binding capacity between heparin oligosaccharides and VIP was analyzed by GMSA and ITC experiments. The binding between heparin oligosaccharides and VIP was simulated using a molecular docking program to show the complex. ELISA assay was used to investigate the effect of non-anticoagulant heparin oligosaccharides on the VIP-mediated cAMP/PKA signaling pathway in vitro. RESULTS: The results indicated that both the length and the sulfation pattern of heparin oligosaccharides affected its binding to VIP. VIP could induce the expression of cAMP at a higher level in PC3 cells, which could be regulated by the interaction of heparin oligosaccharides and VIP. CONCLUSION: The binding between heparin oligosaccharides and VIP could block the binding between VIP and its receptor on tumor cells. Downloading the regulation of the expression level of cAMP could possibly further affect the subsequent activation of PKA. These non-anticoagulant heparin oligosaccharides may block the VIP-mediated cAMP/PKA signaling pathway and thus exert their antitumor activity.

Requirement of microtubules for secretion of a micronemal protein CpTSP4 in the invasive stage of the apicomplexan Cryptosporidium parvum.

The zoonotic Cryptosporidium parvum is a global contributor to infantile diarrheal diseases and opportunistic infections in immunocompromised or weakened individuals. Like other apicomplexans, it possesses several specialized secretory organelles, including micronemes, rhoptry, and dense granules. However, the understanding of cryptosporidial micronemal composition and secretory pathway remains limited. Here, we report a new micronemal protein in C. parvum, namely, thrombospondin (TSP)-repeat domain-containing protein-4 (CpTSP4), providing insights into these ambiguities. Immunostaining and enzyme-linked assays show that CpTSP4 is prestored in the micronemes of unexcysted sporozoites but secreted during sporozoite excystation, gliding, and invasion. In excysted sporozoites, CpTSP4 is also distributed on the two central microtubules unique to Cryptosporidium. The secretion and microtubular distribution could be completely blocked by the selective kinesin-5 inhibitors SB-743921 and SB-715992, resulting in the accumulation of CpTSP4 in micronemes. These support the kinesin-dependent microtubular trafficking of CpTSP4 for secretion. We also localize γ-tubulin, consistent with kinesin-dependent anterograde trafficking. Additionally, recombinant CpTSP4 displays nanomolar binding affinity to the host cell surface, for which heparin acts as one of the host ligands. A novel heparin-binding motif is identified and validated biochemically for its contribution to the adhesive property of CpTSP4 by peptide competition assays and site-directed mutagenesis. These findings shed light on the mechanisms of intracellular trafficking and secretion of a cryptosporidial micronemal protein and the interaction of a TSP-family protein with host cells.IMPORTANCECryptosporidium parvum is a globally distributed apicomplexan parasite infecting humans and/or animals. Like other apicomplexans, it possesses specialized secretory organelles in the zoites, in which micronemes discharge molecules to facilitate the movement and invasion of zoites. Although past and recent studies have identified several proteins in cryptosporidial micronemes, our understanding of the composition, secretory pathways, and domain-ligand interactions of micronemal proteins remains limited. This study identifies a new micronemal protein, namely, CpTSP4, that is discharged during excystation, gliding, and invasion of C. parvum sporozoites. The CpTSP4 secretion depends on the intracellular trafficking on the two Cryptosporidium-unique microtubes that could be blocked by kinesin-5/Eg5 inhibitors. Additionally, a novel heparin-binding motif is identified and biochemically validated, which contributes to the nanomolar binding affinity of CpTSP4 to host cells. These findings indicate that kinesin-dependent microtubular trafficking is critical to CpTSP4 secretion, and heparin/heparan sulfate is one of the ligands for this micronemal protein.