Cell Surface Heparan Sulfate Research Articles

Involvement of Syndecan-1 and Heparanase in Cancer and Inflammation.

The cell surface heparan sulfate proteoglycan Syndecan-1 acts as an important co-receptor for receptor tyrosine kinases and chemokine receptors, and as an adhesion receptor for structural glycoproteins of the extracellular matrix. It serves as a substrate for heparanase, an endo-β-glucuronidase that degrades specific domains of heparan sulfate carbohydrate chains and thereby alters the functional status of the proteoglycan and of Syndecan-1-bound ligands. Syndecan-1 and heparanase show multiple levels of functional interactions, resulting in mutual regulation of their expression, processing, and activity. These interactions are of particular relevance in the context of inflammation and malignant disease. Studies in animal models have revealed a mechanistic role of Syndecan-1 and heparanase in the regulation of contact allergies, kidney inflammation, multiple sclerosis, inflammatory bowel disease, and inflammation-associated tumorigenesis. Moreover, functional interactions between Syndecan-1 and heparanase modulate virtually all steps of tumor progression as defined in the Hallmarks of Cancer. Due to their prognostic value in cancer, and their mechanistic involvement in tumor progression, Syndecan-1 and heparanase have emerged as important drug targets. Data in preclinical models and preclinical phase I/II studies have already yielded promising results that provide a translational perspective.

Changes in heparan sulfate sulfotransferases and cell-surface heparan sulfate during SKM-1 cells granulocytic differentiation and A549 cells epithelial-mesenchymal transition

Heparan sulfate (HS) with various sulfation patterns is one of important modulators of cancer cell fate through interacting with numerous growth factors. Here we found HS 2-O sulfotransferase 1 (HS2ST1) was downregulated at both mRNA and protein levels during granulocytic differentiation of SKM-1 leukemia cells and also HS (glucosamine) 3-O sulfotransferase 3A (HS3ST3A) in epithelial-mesenchymal transition (EMT) of A549 lung cancer cells, meanwhile, cell-surface HS recognized by anti-HS antibody was also changed in both cancer cell lines. Further, HS3ST3A was negatively correlated with the in vitro cell metastasis capability of A549 cells confirmed by RNA interference technology, wound-healing assay and in vitro Matrigel invasion assay. Together, specific HS sulfotransferases may serve as molecular markers and targets for cancer treatment.

3‐O‐Sulfation of Heparan Sulfate Enhances Tau Interaction and Cellular Uptake

AbstractPrion‐like transcellular spreading of tau in Alzheimer's Disease (AD) is mediated by tau binding to cell surface heparan sulfate (HS). However, the structural determinants for tau–HS interaction are not well understood. Microarray and SPR assays of structurally defined HS oligosaccharides show that a rare 3‐O‐sulfation (3‐O‐S) of HS significantly enhances tau binding. In Hs3st1−/− (HS 3‐O‐sulfotransferase‐1 knockout) cells, reduced 3‐O‐S levels of HS diminished both cell surface binding and internalization of tau. In a cell culture, the addition of a 3‐O‐S HS 12‐mer reduced both tau cell surface binding and cellular uptake. NMR titrations mapped 3‐O‐S binding sites to the microtubule binding repeat 2 (R2) and proline‐rich region 2 (PRR2) of tau. Tau is only the seventh protein currently known to recognize HS 3‐O‐sulfation. Our work demonstrates that this rare 3‐O‐sulfation enhances tau–HS binding and likely the transcellular spread of tau, providing a novel target for disease‐modifying treatment of AD and other tauopathies.

3-O-Sulfation of Heparan Sulfate Enhances Tau Interaction and Cellular Uptake.

Prion-like transcellular spreading of tau in Alzheimer's Disease (AD) is mediated by tau binding to cell surface heparan sulfate (HS). However, the structural determinants for tau-HS interaction are not well understood. Microarray and SPR assays of structurally defined HS oligosaccharides show that a rare 3-O-sulfation (3-O-S) of HS significantly enhances tau binding. In Hs3st1-/- (HS 3-O-sulfotransferase-1 knockout) cells, reduced 3-O-S levels of HS diminished both cell surface binding and internalization of tau. In a cell culture, the addition of a 3-O-S HS 12-mer reduced both tau cell surface binding and cellular uptake. NMR titrations mapped 3-O-S binding sites to the microtubule binding repeat 2 (R2) and proline-rich region 2 (PRR2) of tau. Tau is only the seventh protein currently known to recognize HS 3-O-sulfation. Our work demonstrates that this rare 3-O-sulfation enhances tau-HS binding and likely the transcellular spread of tau, providing a novel target for disease-modifying treatment of AD and other tauopathies.

A Pilot Study on Biomarkers of Vascular Injury in Patients with Immune-Mediated Thrombotic Thrombocytopenic Purpura

Background: Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is defined by thrombocytopenia and microangiopathic hemolytic anemia without an alternative explanation, confirmed by severely deficient ADAMTS13 to <10%. It is caused by autoantibodies against the ADAMTS13 protease. Following recovery from an acute iTTP episode, the patient is at risk for relapses and multiple long-term complications including hypertension, depression, headaches and neurocognitive impairment. The etiology of these complications is not well understood. The aim of this study was to identify biomarkers of vascular injury that could help to diagnose or predict the development of these long term complications of iTTP. Four plasma biomarkers of vascular injury were selected with the assistance of a multidisciplinary team of neurologists, pathologists and psychologists, based on their relevance in other chronic diseases and inflammation. Syndecan-1 (CD-138), is a cell surface heparan sulfate proteoglycan that interacts with extracellular matrix molecules and growth factors to maintain epithelial cell morphology. It has been reported to be a negative regulator of various inflammatory processes, with Syndecan-1 knockout (Sdc-1−/−) mice showing enhanced disease severity and impaired recovery.Thrombomodulin (CD141), is an endothelial surface transmembrane glycoprotein. It is involved in the activation of protein C in the inactivation of thrombin. Its expression has been associated with aging and cardiovascular disease.Vascular adhesion protein-1 (VAP) is a member of the copper-containing amine oxidase/semicarbazide-sensitive amine oxidase (AOC/SSAO) enzyme family. It is continuously expressed as a transmembrane glycoprotein in the vascular wall during development and facilitates the accumulation of inflammatory cells into the inflamed environment. It has been shown to be released in cerebral ischemia.E-selectin (CD62E), is a selectin cell adhesion molecule, expressed only in endothelial cells activated by cytokines, and is known to be increased in acute coronary syndrome and atherosclerosis. Methods: Patients with a diagnosis of iTTP functioning normally in their activities of daily living were recruited from existing IRB approved patient cohorts at both the Ohio State University (n = 10) (Columbus) and the University College London Hospitals (n = 15) (London, UK). These patients were studied and reported previously (Cataland, Scully M et al. AJH 2011, 86; 87-89). with a validated measure of neurocognitive function (Cogstate). Patients were characterized by this testing as having normal or abnormal cognitive function. Syndecan-1, thrombomodulin, vascular adhesion protein-1, and E-selectin were studied in plasma samples obtained at the time of the previous neurocognitive testing. All four biomarker assays were performed with commercially available Elisa-based assays. Results: There was a total of 25 patients that underwent neurocognitive testing previously; 11 patients had normal neurocognitive testing and 14 patients were characterized as abnormal. The median age was 41 (range 18-62). One patient was Asian, six were black and 18 were white. Nine patients were male. The demographic information for each group is listed in Table 1. The biomarker results for each group are listed on Table 2 and a comparison is seen on Figure 1. We did see a trend to lower levels of Syndecan-1, and higher levels of thrombomodulin and VAP-1 in patients with abnormal cognitive testing. The two groups had similar levels of CD62E. all biomarker testing was repeated twice and given consistent data. Conclusions: There is a need in the field for a better understanding of the pathophysiology of the long-term complications described in patients with iTTP. This pilot study is the first to look at biomarkers of vascular injury and inflammation that can correlate with neurocognitive impairment and potentially vascular injury in patients with iTTP, not when patients are having an acute episode but during follow up. In addition, we have shown that these biomarkers can be reliably tested in frozen samples from patients with iTPP, and important step for feasibility of future studies. These data are an important first step to develop specific biomarkers that correlate with the development of long-term complications in iTTP patients. Disclosures Masias: Rigel Pharmaceuticals: Consultancy. Scully:Alexion: Consultancy; Novartis: Consultancy; Ablynx/Sanofi: Consultancy; Shire/Takeda: Consultancy; Shire: Research Funding. Cataland:Ablynx/Sanofi: Consultancy, Research Funding; Alexion: Consultancy, Research Funding.

Syndecan-1 and stromal HSPGs: key moderators of communication between myeloma plasma cells and the bone marrow niche

Upon antigen recognition by the B-cell antigen receptor (BCR), B-cells differentiate into plasma cells, which represent the effector cells of the humoral immune system. However, in contrast to their B-cell precursors, plasma no longer express a cell-surface BCR and, hence, are deprived of the BCR-derived signals, which are crucial for survival throughout B-cell development. Instead, for their survival, long-lived plasma cells heavily rely on communication with the bone-marrow (BM) microenvironment, which provides adhesion-mediated as well as multiple soluble signals. Plasma cells acquire strong expression of the cell-surface heparan sulfate proteoglycan (HSPG) syndecan-1, a phenotypic hallmark of plasma cells and their malignant multiple myeloma (MM) counterparts. Our research addresses how syndecan-1, in concert with HSPGs in the bone marrow microenvironment, mediates homing and survival of normal plasma cells, and promotes MM growth, by co-opting growth an survival factors from the BM niche and targeting them to their cognate receptors. The crucial function of syndecan-1 and of stromal HSPGs in the communication of MM with the bone marrow niche, designates these molecules and their synthesis machinery as potential treatment targets. Various venues to target syndecan-1/HSPGs in MM are currently being explored in preclinical and clinical studies. van Andel et al. PNAS 114:376-381 (2017). Ren et al. Blood 131:982-994 (2018).

Comparison of the Interactions of Different Growth Factors and Glycosaminoglycans.

Most growth factors are naturally occurring proteins, which are signaling molecules implicated in cellular multiple functions such as proliferation, migration and differentiation under patho/physiological conditions by interacting with cell surface receptors and other ligands in the extracellular microenvironment. Many of the growth factors are heparin-binding proteins (HBPs) that have a high affinity for cell surface heparan sulfate proteoglycans (HSPG). In the present study, we report the binding kinetics and affinity of heparin interacting with different growth factors, including fibroblast growth factor (FGF) 2,7,10, hepatocyte growth factor (HGF) and transforming growth factor (TGF β-1), using a heparin chip. Surface plasmon resonance studies revealed that all the tested growth factors bind to heparin with high affinity (with KD ranging from ~0.1 to 59 nM) and all the interactions are oligosaccharide size dependent except those involving TGF β-1. These heparin-binding growth factors also interact with other glycosaminoglycans (GAGs), as well as various chemically modified heparins. Other GAGs, including heparan sulfate, chondroitin sulfates A, B, C, D, E and keratan sulfate, showed different inhibition activities for the growth factor-heparin interactions. FGF2, FGF7, FGF10 and HGF bind heparin but the 2-O-sulfo and 6-O-sulfo groups on heparin have less impact on these interactions than do the N-sulfo groups. All the three sulfo groups (N-, 2-O and 6-O) on heparin are important for TGFβ-1-heparin interaction.

The melanocortin-1 receptor reversely regulates the melanin synthesis and migration of melanoma cells via dimerization-induced conformational changes

We previously reported that the melanocortin-1 receptor (MC1R), a key regulator of melanogenesis, regulates cell migration; however, the detailed mechanism remained unknown. Since the homo-dimerization of MC1R by four inter-subunit disulfide bonds is known to be functionally important for melanogenesis, we investigated the importance of MC1R dimerization for cell migration. Unlike the wild-type MC1R, the dimerization-defective mutant MC1R in which four critical Cys residues were replaced with Ala residues (Cys35-267-273-275Ala) significantly inhibited melanin synthesis but enhanced cell migration in human MNT-1 and A375 melanoma cells. This suggests that there may be a reverse correlation between melanin synthesis and cell migration. Interestingly, melanoma cells expressing the dimerization-defective mutant exhibited enhanced expression of the cell surface heparan sulfate proteoglycan, syndecan-2, and knockdown of syndecan-2 expression decreased the mutant-mediated cell migration. Consistently, ASIP, an antagonist of MC1R, enhanced syndecan-2 expression and cell migration and reversed the α-melanocyte-stimulating hormone (α-MSH)-mediated inhibition of syndecan-2 expression. Furthermore, α-MSH reduced the cell migration of MNT1 cells expressing wild-type MC1R but not its dimerization-defective mutant. Together, these data strongly suggest that MC1R reversely regulates melanin synthesis and migration via the conformational changes induced by dimerization.

Abstract 2309: Chimeric antigen receptor T-cell therapy targeting glypican-1 in pancreatic cancer

Abstract Pancreatic cancer is the fourth most common cause of death from cancer in the US, with a 5-year overall survival of 8.5%. While immunotherapy with chimeric antigen receptor (CAR) T cells has shown promise in certain hematological malignancies, their efficacy for solid tumors including pancreatic cancer remains elusive. Thus, laboratory research is urgently needed to identify and validate a new target of CAR T-cell therapy for patients with pancreatic cancer. In this study, we made CAR T cells that recognize glypican-1 (GPC1), a cell surface heparan sulfate proteoglycan protein that is overexpressed in pancreatic cancer. We isolated two GPC1-specific antibodies; the mouse monoclonal antibody HM2 that binds the C-lobe of GPC1 close to the cell surface, and the camel single domain antibody D4 that recognizes a conformational epitope in the protein core of GPC1. The transduction efficiencies of CAR (HM2) and CAR (D4) in primary T cells were 86% and 58%, respectively. GPC1-overexpressing KLM1 cells (2B9) and GPC1-overexpressing A431 cells (H8) were efficiently lysed by both CAR T cells in dose-dependent manners. By contrast, minimal cell lysis was observed in GPC1-negative cell line A431. In the presence of GPC1-positive tumor cells, greater amount of TNF-α, IFN-γ and IL-2 were produced by GPC1-specific CAR T cells than mock-transduced T cells. Although both CAR T cells showed similar cytolytic activity in vitro, CAR (D4) T cells induced 2-4 times more cytokines than CAR (HM2) T cells when co-cultured with GPC1-postive tumor cells. Notably, both GPC1-targeted CAR T cells demonstrated potent antitumor efficacy in a peritoneal dissemination xenograft mouse model. Our findings indicate that GPC1-targeted CAR T cell therapy could be a new approach for treating pancreatic cancer. Citation Format: Nan Li, Dan Li, Hua Ren, Madeline Torres, Mitchell Ho. Chimeric antigen receptor T-cell therapy targeting glypican-1 in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2309.

Abstract 1913: Role of glypican 1 in regulating fibroblast activation in the prostate cancer tumor microenvironment

Abstract Cancer is a heterogeneous collection of neoplastic cells, surrounded by non-malignant, stromal cells, and extracellular matrix (ECM), among other constituents. Cross-talk between tumor and stromal cells forms a dynamic tumor microenvironment (TME) that mediates cell growth, proliferation, and metastasis. The TME can inhibit cancer growth and proliferation during the initial stages of carcinogenesis; however, cancer cells can evade these inhibitory signals, and reshape the TME, making it more amenable to their own growth. This gives rise to cancer-associated stromal cells, including cancer-associated fibroblasts (CAFs). CAFs further modulate the TME by regulating the release of growth signals, cell death, ECM remodeling, angiogenesis, and epithelial-mesenchymal transition. Glypican-1 (GPC1) is a cell surface heparan sulfate proteoglycan (HSPG) that has both pro-mitogenic and pro-metastatic activity. GPC1’s role in regulating fibroblasts and their transformation into CAFs has not been investigated. We studied the ability of prostate cancer cells to alter the TME in vitro by exposing human bone fibroblasts (HS-5 cells) to tumor conditioned media (TCM) isolated from a human neuroendocrine (DU-145) and bone (PC-3) metastatic prostate cancer cell lines and assessed changes in cell morphology and cytotoxicity (using MTT). Exposure of HS-5 cells to TCM from DU-145 and PC-3 cells altered cellular morphology to a more mesenchymal phenotype similar to that seen in active myofibroblasts. TCM also decreased MTT staining in a time-dependent manner. We also determined the effect of GPC1 knockdown (KD) in HS-5 cells on cell morphology, migration and cytotoxicity. Interestingly, we saw phenotypic switching of GPC1 KD HS-5 cells to a myofibroblast like morphology akin to that when treated with TCM. There was no change in cytotoxicity in GPC1 KD HS-5 cells as compared to control cells. However, GPC1 KD enhanced the migration of HS-5 cells in wound healing assay. Migration was also increased in GPC1 KD HS-5 cells upon treatment with TCM. GPC1 KD also increased HS-5 cell contractility significantly. Collectively, these data support the hypothesis that GPC1 plays an inhibitory role in the TME of prostate cancer by regulating the activation of fibroblasts. Citation Format: Sukhneeraj P. Kaur, Hee K. Lee, Robert D. Arnold, Brian S. Cummings. Role of glypican 1 in regulating fibroblast activation in the prostate cancer tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1913.

Abstract 3445: Mechanism, physiological and therapeutic implications of LGR-independent potentiation of WNT signaling by R-spondins

Abstract The WNT signaling pathway encodes positional information in animals, orchestrates patterning and morphogenesis during embryonic development, and promotes tissue renewal and regeneration in adults. Dysregulation of WNT signaling has been implicated in many diseases, including developmental malformations, skeletal and dental abnormalities, cardiovascular and neurodegenerative disorders, diabetes, and many types of cancer. Members of the R-spondin family of secreted growth factors have emerged as key regulators of WNT signaling strength. The R-spondin system is a vertebrate adaptation that may be related to the appearance of sophisticated stem cell compartments, which require exquisite control of WNT signaling. The four members of the family (R-spondin 1-4) can strongly potentiate responses to WNT ligands during development and in stem cells, but the mechanisms by which they transduce signals are not fully understood and the reasons why they produce different physiological effects are largely unknown. Unexpectedly, we discovered that R-spondins 2 and 3 can uniquely potentiate WNT signaling in cells lacking LGRs 4, 5 and 6, the principal R-spondin receptors. We determined the protein domains on R-spondins necessary and sufficient for this new mode of signaling, and showed that it is mediated by an alternative interaction with cell-surface heparan sulfate proteoglycans. This finding is transformative because LGRs 4-6 were thought to be required to transduce all R-spondin signals and hence determine their site of action. Indeed, these LGRs are highly expressed in many stem cell compartments whose maintenance depends on potentiation of WNT signaling by R-spondins. Instead our work shows that there are two modes of signaling by R-spondins, an LGR-dependent and an LGR-independent mode, and that different R-spondins may use distinct molecular mechanisms to transduce signals in various biological contexts. Supporting our findings, recent work from another group demonstrated that during limb development, R-spondin 2 signals through an LGR-independent mechanism. Since components of the R-spondin signaling module are commonly mutated in cancer, therapeutic targeting of LGR-independent signaling deserves evaluation. I will present the results demonstrating our conclusions and discuss their implications in physiology and disease. Citation Format: Andres M. Lebensohn, Rajat Rohatgi. Mechanism, physiological and therapeutic implications of LGR-independent potentiation of WNT signaling by R-spondins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3445.

Ranaviruses Bind Cells from Different Species through Interaction with Heparan Sulfate.

Ranavirus cross-species infections have been documented, but the viral proteins involved in the interaction with cell receptors have not yet been identified. Here, viral cell-binding proteins and their cognate cellular receptors were investigated using two ranaviruses, Andrias davidianus ranavirus (ADRV) and Rana grylio virus (RGV), and two different cell lines, Chinese giant salamander thymus cells (GSTC) and Epithelioma papulosum cyprinid (EPC) cells. The heparan sulfate (HS) analog heparin inhibited plaque formation of ADRV and RGV in the two cell lines by more than 80% at a concentration of 5 μg/mL. In addition, enzymatic removal of cell surface HS by heparinase I markedly reduced plaque formation by both viruses and competition with heparin reduced virus-cell binding. These results indicate that cell surface HS is involved in ADRV and RGV cell binding and infection. Furthermore, recombinant viral envelope proteins ADRV-58L and RGV-53R bound heparin-Sepharose beads implying the potential that cell surface HS is involved in the initial interaction between ranaviruses and susceptible host cells. To our knowledge, this is the first report identifying cell surface HS as ranavirus binding factor and furthers understanding of interactions between ranaviruses and host cells.

Proteoglycans and glycosaminoglycans as regulators of cancer stem cell function and therapeutic resistance.

In contrast to the bulk of the tumor, a subset of cancer cells called cancer stem cells (CSC; or tumor-initiating cells) is characterized by self-renewal, unlimited proliferative potential, expression of multidrug resistance proteins, active DNA repair capacity, apoptosis resistance, and a considerable developmental plasticity. Due to these properties, CSCs display increased resistance to chemo- and radiotherapy. Recent findings indicate that aberrant functions of proteoglycans (PGs) and glycosaminoglycans (GAGs) contribute substantially to the CSC phenotype and therapeutic resistance. In this review, we summarize how the diverse functions of the glycoproteins and carbohydrates facilitate acquisition and maintenance of the CSC phenotype, and how this knowledge can be exploited to develop novel anticancer therapies. For example, the large transmembrane chondroitin sulfate PG NG2/CSPG4 marks stem cell (SC) populations in brain tumors. Cell surface heparan sulfate PGs of the syndecan and glypican families modulate the stemness-associated Wnt, hedgehog, and notch signaling pathways, whereas the interplay of hyaluronan in the SC niche with CSC CD44 determines the maintenance of stemness and promotes therapeutic resistance. A better understanding of the molecular mechanisms by which PGs and GAGs regulate CSC function will aid the development of targeted therapeutic approaches which could avoid relapse after an otherwise successful conventional therapy. Chimeric antigen receptor T cells, PG-primed dendritic cells, PG-targeted antibody-drug conjugates, and inhibitory peptides and glycans have already shown highly promising results in preclinical models.

Pathogenic Variants in GPC4 Cause Keipert Syndrome

Glypicans are a family of cell-surface heparan sulfate proteoglycans that regulate growth-factor signaling during development and are thought to play a role in the regulation of morphogenesis. Whole-exome sequencing of the Australian family that defined Keipert syndrome (nasodigitoacoustic syndrome) identified a hemizygous truncating variant in the gene encoding glypican 4 (GPC4). This variant, located in the final exon of GPC4, results in premature termination of the protein 51 amino acid residues prior to the stop codon, and in concomitant loss of functionally important N-linked glycosylation (Asn514) and glycosylphosphatidylinositol (GPI) anchor (Ser529) sites. We subsequently identified seven affected males from five additional kindreds with novel and predicted pathogenic variants in GPC4. Segregation analysis and X-inactivation studies in carrier females provided supportive evidence that the GPC4 variants caused the condition. Furthermore, functional studies of recombinant protein suggested that the truncated proteins p.Gln506∗ and p.Glu496∗ were less stable than the wild type. Clinical features of Keipert syndrome included a prominent forehead, a flat midface, hypertelorism, a broad nose, downturned corners of mouth, and digital abnormalities, whereas cognitive impairment and deafness were variable features. Studies of Gpc4 knockout mice showed evidence of the two primary features of Keipert syndrome: craniofacial abnormalities and digital abnormalities. Phylogenetic analysis demonstrated that GPC4 is most closely related to GPC6, which is associated with a bone dysplasia that has a phenotypic overlap with Keipert syndrome. Overall, we have shown that pathogenic variants in GPC4 cause a loss of function that results in Keipert syndrome, making GPC4 the third human glypican to be linked to a genetic syndrome.

Glycoprotein K8.1A of Kaposi's Sarcoma-Associated Herpesvirus Is a Critical B Cell Tropism Determinant Independent of Its Heparan Sulfate Binding Activity.

B lymphocytes are the major cellular reservoir in individuals infected with Kaposi's sarcoma-associated herpesvirus (KSHV), and the virus is etiologically linked to two B cell lymphoproliferative disorders. We previously described the MC116 human B cell line as a KSHV-susceptible model to overcome the paradoxical refractoriness of B cell lines to experimental KSHV infection. Here, using monoclonal antibody inhibition and a deletion mutant virus, we demonstrate that the KSHV virion glycoprotein K8.1A is critical for infection of MC116, as well as tonsillar B cells; in contrast, we confirm previous reports on the dispensability of the glycoprotein for infection of primary endothelial cells and other commonly studied non-B cell targets. Surprisingly, we found that the role of K8.1A in B cell infection is independent of its only known biochemical activity of binding to surface heparan sulfate, suggesting the possible involvement of an additional molecular interaction(s). Our finding that K8.1A is a critical determinant for KSHV B cell tropism parallels the importance of proteins encoded by positionally homologous genes for the cell tropism of other gammaherpesviruses.IMPORTANCE Elucidating the molecular mechanisms by which KSHV infects B lymphocytes is critical for understanding how the virus establishes lifelong persistence in infected people, in whom it can cause life-threatening B cell lymphoproliferative disease. Here, we show that K8.1A, a KSHV-encoded glycoprotein on the surfaces of the virus particles, is critical for infection of B cells. This finding stands in marked contrast to previous studies with non-B lymphoid cell types, for which K8.1A is known to be dispensable. We also show that the required function of K8.1A in B cell infection does not involve its binding to cell surface heparan sulfate, the only known biochemical activity of the glycoprotein. The discovery of this critical role of K8.1A in KSHV B cell tropism opens promising new avenues to unravel the complex mechanisms underlying infection and disease caused by this viral human pathogen.

Long term safety of targeted internalization of cell penetrating peptide crotamine into renal proximal tubular epithelial cells in vivo

Activated proximal tubular epithelial cells (PTECs) play a crucial role in progressive tubulo-interstitial fibrosis in native and transplanted kidneys. Targeting PTECs by non-viral delivery vectors might be useful to influence the expression of important genes and/or proteins in order to slow down renal function loss. However, no clinical therapies that specifically target PTECs are available at present. We earlier showed that a cationic cell penetrating peptide isolated from South American rattlesnake venom, named crotamine, recognizes cell surface heparan sulfate proteoglycans and accumulates in cells. In healthy mice, crotamine accumulates mainly in kidneys after intraperitoneal (ip) injection. Herein we demonstrate for the first time, the overall safety of acute or long-term treatment with daily ip administrated crotamine for kidneys functions. Accumulation of ip injected crotamine in the kidney brush border zone of PTECs, and its presence inside these cells were observed. In addition, significant lower in vitro crotamine binding, uptake and reporter gene transport and expression could be observed in syndecan-1 deficient HK-2 PTECs compared to wild-type cells, indicating that the absence of syndecan-1 impairs crotamine uptake into PTECs. Taken together, our present data show the safety of in vivo long-term treatment with crotamine, and its preferential uptake into PTECs, which are especially rich in HSPGs such as syndecan-1. In addition to the demonstrated in vitro gene delivery mediated by crotamine in HK-2 cells, the potential applicability of crotamine as prototypic non-viral (gene) delivery nanocarrier to modulate PTEC gene and/or protein expression was confirmed.

The Selection of a Hepatocyte Cell Line Susceptible to Plasmodium falciparum Sporozoite Invasion That Is Associated With Expression of Glypican-3.

In vitro studies of liver stage (LS) development of the human malaria parasite Plasmodium falciparum are technically challenging; therefore, fundamental questions about hepatocyte receptors for invasion that can be targeted to prevent infection remain unanswered. To identify novel receptors and to further understand human hepatocyte susceptibility to P. falciparum sporozoite invasion, we created an optimized in vitro system by mimicking in vivo liver conditions and using the subcloned HC-04.J7 cell line that supports mean infection rates of 3–5% and early development of P. falciparum exoerythrocytic forms—a 3- to 5-fold improvement on current in vitro hepatocarcinoma models for P. falciparum invasion. We juxtaposed this invasion-susceptible cell line with an invasion-resistant cell line (HepG2) and performed comparative proteomics and RNA-seq analyses to identify host cell surface molecules and pathways important for sporozoite invasion of host cells. We identified and investigated a hepatocyte cell surface heparan sulfate proteoglycan, glypican-3, as a putative mediator of sporozoite invasion. We also noted the involvement of pathways that implicate the importance of the metabolic state of the hepatocyte in supporting LS development. Our study highlights important features of hepatocyte biology, and specifically the potential role of glypican-3, in mediating P. falciparum sporozoite invasion. Additionally, it establishes a simple in vitro system to study the LS with improved invasion efficiency. This work paves the way for the greater malaria and liver biology communities to explore fundamental questions of hepatocyte-pathogen interactions and extend the system to other human malaria parasite species, like P. vivax.

Glypican-1 Promotes Tumorigenesis by Regulating the PTEN/Akt/β-Catenin Signaling Pathway in Esophageal Squamous Cell Carcinoma.

Glypican-1 (GPC1), a cell-surface heparan sulfate proteoglycan, promotes the pathogenesis of many human cancers. This study focuses on the role of GPC1 in the promotion of cell proliferation and motility in esophageal squamous cell carcinoma (ESCC). The expression and distribution of GPC1 were measured in tumor tissues from 248 ESCC patients using immunohistochemical (IHC) assays. Cell counting (kit-8), flow cytometry, Transwell, wound healing, IHC, and Western blotting assays were performed to examine the molecular mechanisms that underlie how GPC1 enhances cell proliferation and motility. The level of GPC1 was higher in ESCC tumor samples than in para-tumor tissues (IHC score: 5.42 ± 2.15 vs. 0.86 ± 0.96). Ectopic overexpression of GPC1 in EC9706 cells promoted cell growth and the G1/S phase transition; conversely, GPC1 knockdown in Eca109 cells attenuated cell proliferation and induced G2/M phase arrest. In addition, GPC1 upregulation enhanced ESCC cell motility and induced epithelial mesenchymal transition (EMT), as demonstrated by the aberrant expression of EMT markers. Mechanistically, we demonstrated that GPC1 increased levels of p-Akt and β-catenin and reduced PTEN expression in ESCC. Our study indicated that GPC1 promotes the aggressive proliferation of ESCC cells by regulating the PTEN/Akt/β-catenin pathway. GPC1 may be a promising target for ESCC treatment.

Contribution of syndecans to cellular internalization and fibrillation of amyloid-\u03b2(1\u201342)

Intraneuronal accumulation of amyloid-β(1–42) (Aβ1–42) is one of the earliest signs of Alzheimer’s disease (AD). Cell surface heparan sulfate proteoglycans (HSPGs) have profound influence on the cellular uptake of Aβ1–42 by mediating its attachment and subsequent internalization into the cells. Colocalization of amyloid plaques with members of the syndecan family of HSPGs, along with the increased expression of syndecan-3 and -4 have already been reported in postmortem AD brains. Considering the growing evidence on the involvement of syndecans in the pathogenesis of AD, we analyzed the contribution of syndecans to cellular uptake and fibrillation of Aβ1–42. Among syndecans, the neuron specific syndecan-3 isoform increased cellular uptake of Aβ1–42 the most. Kinetics of Aβ1–42 uptake also proved to be fairly different among SDC family members: syndecan-3 increased Aβ1–42 uptake from the earliest time points, while other syndecans facilitated Aβ1–42 internalization at a slower pace. Internalized Aβ1–42 colocalized with syndecans and flotillins, highlighting the role of lipid-rafts in syndecan-mediated uptake. Syndecan-3 and 4 also triggered fibrillation of Aβ1–42, further emphasizing the pathophysiological relevance of syndecans in plaque formation. Overall our data highlight syndecans, especially the neuron-specific syndecan-3 isoform, as important players in amyloid pathology and show that syndecans, regardless of cell type, facilitate key molecular events in neurodegeneration.

Fibroblast Growth Factor-2 Binding to Heparan Sulfate Proteoglycans Varies with Shear Stress in Flow-Adapted Cells.

Fibroblast growth factor 2 (FGF2), an important regulator of angiogenesis, binds to endothelial cell (EC) surface FGF receptors (FGFRs) and heparan sulfate proteoglycans (HSPGs). FGF2 binding kinetics have been predominantly studied in static culture; however, the endothelium is constantly exposed to flow which may affect FGF2 binding. We therefore used experimental and computational techniques to study how EC FGF2 binding changes in flow. ECs adapted to 24h of flow demonstrated biphasic FGF2-HSPG binding, with FGF2-HSPG complexes increasing up to 20dynes/cm2 shear stress and then decreasing at higher shear stresses. To understand how adaptive EC surface remodeling in response to shear stress may affect FGF2 binding to FGFR and HSPG, we implemented a computational model to predict the relative effects of flow-induced surface receptor changes. We then fit the computational model to the experimental data using relationships between HSPG availability and FGF2-HSPG dissociation and flow that were developed from a basement membrane study, as well as including HSPG production. These studies suggest that FGF2 binding kinetics are altered in flow-adapted ECs due to changes in cell surface receptor quantity, availability, and binding kinetics, which may affect cell growth factor response.