Cell-surface proteoglycans as molecular portals for cationic peptide and polymer entry into cells

Polycationic macromolecules and cationic peptides acting as PTDs (protein transduction domains) and CPPs (cell-penetrating peptides) represent important classes of agents used for the import and delivery of a wide range of molecular cargoes into cells. Their entry into cells is typically initiated through interaction with cell-surface HS (heparan sulfate) molecules via electrostatic interactions, followed by endocytosis of the resulting complexes. However, the endocytic mechanism employed (clathrin-mediated endocytosis, caveolar uptake or macropinocytosis), defining the migration of these peptides into cells, depends on parameters such as the nature of the cationic agent itself and complex formation with cargo, as well as the nature and distribution of proteoglycans expressed on the cell surface. Moreover, a survey of the literature suggests that endocytic pathways should not be considered as mutually exclusive, as more than one entry mechanism may be operational for a given cationic complex in a particular cell type. Specifically, the observed import may best be explained by the distribution and uptake of cell-surface HSPGs (heparan sulfate proteoglycans), such as syndecans and glypicans, which have been shown to mediate the uptake of many ligands besides cationic polymers. A brief overview of the roles of HSPGs in ligand internalization is presented, as well as mechanistic hypotheses based on the known properties of these cell-surface markers. The identification and investigation of interactions made by glycosaminoglycans and core proteins of HSPGs with PTDs and cationic polymers will be crucial in defining their uptake by cells.

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Neuropilin-1 and heparan sulfate proteoglycans cooperate in cellular uptake of nanoparticles functionalized by cationic cell-penetrating peptides

Science Advances ◽

10.1126/sciadv.1500821 ◽

2015 ◽

Vol 1 (10) ◽

pp. e1500821 ◽

Cited By ~ 36

Author(s):

Hong-Bo Pang ◽

Gary B. Braun ◽

Erkki Ruoslahti

Keyword(s):

Cell Surface ◽

Heparan Sulfate ◽

Vascular Permeability ◽

Mammalian Cells ◽

Cell Penetrating Peptides ◽

Proteolytic Processing ◽

Cell Surface Receptor ◽

Neuropilin 1 ◽

Cell Penetrating ◽

Surface Receptor

Cell-penetrating peptides (CPPs) have been widely used to deliver nanomaterials and other types of macromolecules into mammalian cells for therapeutic and diagnostic use. Cationic CPPs that bind to heparan sulfate (HS) proteoglycans on the cell surface induce potent endocytosis; however, the role of other surface receptors in this process is unclear. We describe the convergence of an HS-dependent pathway with the C-end rule (CendR) mechanism that enables peptide ligation with neuropilin-1 (NRP1), a cell surface receptor known to be involved in angiogenesis and vascular permeability. NRP1 binds peptides carrying a positive residue at the carboxyl terminus, a feature that is compatible with cationic CPPs, either intact or after proteolytic processing. We used CPP and CendR peptides, as well as HS- and NRP1-binding motifs from semaphorins, to explore the commonalities and differences of the HS and NRP1 pathways. We show that the CendR-NRP1 interaction determines the ability of CPPs to induce vascular permeability. We also show at the ultrastructural level, using a novel cell entry synchronization method, that both the HS and NRP1 pathways can initiate a macropinocytosis-like process and visualize these CPP-cargo complexes going through various endosomal compartments. Our results provide new insights into how CPPs exploit multiple surface receptor pathways for intracellular delivery.

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Membrane anchoring of heparan sulfate proteoglycans by phosphatidylinositol and kinetics of synthesis of peripheral and detergent-solubilized proteoglycans in Schwann cells.

The Journal of Cell Biology ◽

10.1083/jcb.108.5.1891 ◽

1989 ◽

Vol 108 (5) ◽

pp. 1891-1897 ◽

Cited By ~ 44

Author(s):

D J Carey ◽

D M Evans

Keyword(s):

Cell Surface ◽

Heparan Sulfate ◽

Schwann Cells ◽

Electrostatic Interactions ◽

Heparan Sulfate Proteoglycans ◽

Membrane Bound ◽

Covalently Linked ◽

Mode Of Attachment ◽

Membrane Anchoring ◽

Kinetics Of

Previous studies have shown that Schwann cells synthesize both peripheral and integral hydrophobic cell surface heparan sulfate proteoglycans (HSPGs). The experiments reported here were undertaken to investigate the mode of attachment of these proteins to the cell surface and their potential interrelationship. The binding of the hydrophobic HSPGs to membranes appears to be via covalently linked phosphatidylinositol based on the observation that incubation of the detergent-solubilized protein with purified phosphatidylinositol-specific phospholipase C significantly reduces the ability of the HSPGs to associate with phospholipid vesicles in a reconstitution assay. The peripherally associated HSPGs were released from the cells by incubation in the presence of heparin (10 mg/ml), 10 mM phytic acid (inositol hexaphosphate), or 2 M NaCl. These treatments also solubilized basement membrane HSPGs synthesized by the Schwann cells. These data suggest that the peripheral HSPGs are bound to the surface by electrostatic interactions. The peripheral and hydrophobic HSPGs were identical in overall size, net charge, length of glycosaminoglycan chains, and patterns of N-sulfation. To determine whether the peripheral HSPGs were derived from the membrane-bound form by cleavage of the membrane anchor, we examined the kinetics of synthesis and degradation of the two forms of HSPGs. The results obtained indicated the existence of two pools of detergent-solubilized HSPG with fast (t1/2 = 6 h) and slow (t1/2 = 55 h) turnover kinetics. The data were consistent with a model in which the peripheral HSPGs were derived from the slowly turning over pool of detergent-solubilized HSPGs.

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An investigation of genetic polymorphisms in Heparan Sulfate Proteoglycan core proteins and key modification enzymes in an Australian Caucasian multiple sclerosis population

10.21203/rs.2.23090/v1 ◽

2020 ◽

Author(s):

Rachel K Okolicsanyi ◽

Julia Bluhm ◽

Cassandra Miller ◽

Lyn R Griffiths ◽

Larisa M Haupt

Keyword(s):

Multiple Sclerosis ◽

Cell Surface ◽

Heparan Sulfate ◽

Demyelinating Disease ◽

Core Protein ◽

Association Studies ◽

International Studies ◽

Genome Wide Association Studies ◽

Nucleotide Polymorphisms ◽

Core Proteins

Abstract Multiple Sclerosis (MS) is a chronic inflammatory demyelinating disease affecting the central nervous system in young adults. Heparan sulfate proteoglycans (HSPGs) are ubiquitous to the cell surface and the extracellular matrix. HSPG biosynthesis is a complex process involving enzymatic attachment of heparan sulfate (HS) chains to a core protein. HS side chains mediate specific ligand and growth factor interactions directing cellular processes including cell adhesion, migration and differentiation. Two main families of HSPGs exist, the syndecans (SDC1-4) and glypicans (GPC1-6). The SDCs are transmembrane proteins, while the GPC family are GPI-linked to the cell surface. SDC1 has well-documented interactions with numerous signalling pathways. Genome wide association studies (GWAS) have identified regions of the genome associated with MS including a region on chromosome 13 containing GPC5 and GPC6. International studies have revealed significant associations between this region and disease development. Exostosin-1 (EXT1) and sulfatase-1 (SULF1) are two enzymes responsible for the generation of HS chains. EXT1, with documented tumour suppressor properties, is involved in initiation and polymerisation of the growing HS chain. SULF1 removes 6- O -sulfate groups from HS chains, thereby affecting protein-ligand interactions and subsequent downstream signalling with HS modification potentially having significant effects on MS progression. In this study we identified significant associations between single nucleotide polymorphisms in SDC1, GPC5 and GPC6 and MS in an Australian Caucasian case control population. Further significant associations in these genes were identified when the population was stratified by sex and disease subtype. No association was found for EXT1 or SULF1.

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Alveolar type II cells synthesize hydrophobic cell-associated proteoglycans with multiple core proteins

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1992.263.3.l348 ◽

1992 ◽

Vol 263 (3) ◽

pp. L348-L356 ◽

Cited By ~ 2

Author(s):

W. M. Maniscalco ◽

M. H. Campbell

Keyword(s):

Extracellular Matrix ◽

Cell Surface ◽

Heparan Sulfate ◽

Core Protein ◽

Primary Cultures ◽

Alveolar Type ◽

Type Ii Cells ◽

Type Ii ◽

Alveolar Type Ii Cells ◽

Core Proteins

Type II alveolar epithelial cells interact with the extracellular matrix via cell surface receptors for matrix ligands. Cell surface proteoglycans, which are hydrophobic due to their membrane insertion domains, are one of several classes of molecules that may be receptors for matrix ligands. To analyze the hydrophobic proteoglycans synthesized by adult alveolar type II cells, we labeled these cells with 35SO4 and [3H]leucine in short-term primary cultures. Cell-associated hydrophobic proteoglycans and culture medium-derived proteoglycans were purified and characterized. Both the hydrophobic proteoglycans and medium-derived proteoglycans, which were not hydrophobic, had mainly heparan sulfate glycosaminoglycans. Analysis of core proteins of the hydrophobic proteoglycans showed three proteins, 47, 65, and 90 kDa. The 47- and 65-kDa core proteins were substituted only with heparan sulfate chains. The 90-kDa core protein was seen only after digestion with both heparitinase and chondroitin ABC lyase, suggesting it was a hybrid having both heparan sulfate and chondroitin-dermatan sulfate chains. These findings were confirmed by iodination of the core proteins. The hydrophobic cell-associated proteoglycans inserted into artificial liposomes, whereas the medium-derived molecules did not. These data document heterogeneity in core protein and glycosaminoglycan chains among hydrophobic proteoglycans synthesized in vitro by adult alveolar type II cells. These molecules may have diverse functions in regulating type II cell interaction with the extracellular matrix.

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Shedding of Syndecan-1 and -4 Ectodomains Is Regulated by Multiple Signaling Pathways and Mediated by a Timp-3–Sensitive Metalloproteinase

The Journal of Cell Biology ◽

10.1083/jcb.148.4.811 ◽

2000 ◽

Vol 148 (4) ◽

pp. 811-824 ◽

Cited By ~ 278

Author(s):

Marilyn L. Fitzgerald ◽

Zihua Wang ◽

Pyong Woo Park ◽

Gillian Murphy ◽

Merton Bernfield

Keyword(s):

Cell Surface ◽

Heparan Sulfate ◽

Signaling Pathways ◽

Proteolytic Activity ◽

Intracellular Signaling ◽

Protein Tyrosine Kinase ◽

Core Protein ◽

Ectodomain Shedding ◽

Core Proteins

The syndecan family of four transmembrane heparan sulfate proteoglycans binds a variety of soluble and insoluble extracellular effectors. Syndecan extracellular domains (ectodomains) can be shed intact by proteolytic cleavage of their core proteins, yielding soluble proteoglycans that retain the binding properties of their cell surface precursors. Shedding is accelerated by PMA activation of protein kinase C, and by ligand activation of the thrombin (G-protein–coupled) and EGF (protein tyrosine kinase) receptors (Subramanian, S.V., M.L. Fitzgerald, and M. Bernfield. 1997. J. Biol. Chem. 272:14713–14720). Syndecan-1 and -4 ectodomains are found in acute dermal wound fluids, where they regulate growth factor activity (Kato, M., H. Wang, V. Kainulainen, M.L. Fitzgerald, S. Ledbetter, D.M. Ornitz, and M. Bernfield. 1998. Nat. Med. 4:691–697) and proteolytic balance (Kainulainen, V., H. Wang, C. Schick, and M. Bernfield. 1998. J. Biol. Chem. 273:11563–11569). However, little is known about how syndecan ectodomain shedding is regulated. To elucidate the mechanisms that regulate syndecan shedding, we analyzed several features of the process that sheds the syndecan-1 and -4 ectodomains. We find that shedding accelerated by various physiologic agents involves activation of distinct intracellular signaling pathways; and the proteolytic activity responsible for cleavage of syndecan core proteins, which is associated with the cell surface, can act on unstimulated adjacent cells, and is specifically inhibited by TIMP-3, a matrix-associated metalloproteinase inhibitor. In addition, we find that the syndecan-1 core protein is cleaved on the cell surface at a juxtamembrane site; and the proteolytic activity responsible for accelerated shedding differs from that involved in constitutive shedding of the syndecan ectodomains. These results demonstrate the existence of highly regulated mechanisms that can rapidly convert syndecans from cell surface receptors or coreceptors to soluble heparan sulfate proteoglycan effectors. Because the shed ectodomains are found and function in vivo, regulation of syndecan ectodomain shedding by physiological mediators indicates that shedding is a response to specific developmental and pathophysiological cues.

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Heparan Sulfate Proteoglycans Are Required for Cellular Binding of the Hepatitis E Virus ORF2 Capsid Protein and for Viral Infection

Journal of Virology ◽

10.1128/jvi.00717-09 ◽

2009 ◽

Vol 83 (24) ◽

pp. 12714-12724 ◽

Cited By ~ 119

Author(s):

Manjula Kalia ◽

Vivek Chandra ◽

Sheikh Abdul Rahman ◽

Deepak Sehgal ◽

Shahid Jameel

Keyword(s):

Cell Surface ◽

Heparan Sulfate ◽

Capsid Protein ◽

Marked Reduction ◽

Electrostatic Interactions ◽

Hepatitis E Virus ◽

Hepatitis E ◽

Heparan Sulfate Proteoglycans ◽

Productive Infection ◽

Target Cells

ABSTRACT The hepatitis E virus (HEV), a nonenveloped RNA virus, is the causative agent of hepatitis E. The mode by which HEV attaches to and enters into target cells for productive infection remains unidentified. Open reading frame 2 (ORF2) of HEV encodes its major capsid protein, pORF2, which is likely to have the determinants for virus attachment and entry. Using an ∼56-kDa recombinant pORF2 that can self-assemble as virus-like particles, we demonstrated that cell surface heparan sulfate proteoglycans (HSPGs), specifically syndecans, play a crucial role in the binding of pORF2 to Huh-7 liver cells. Removal of cell surface heparan sulfate by enzymatic (heparinase) or chemical (sodium chlorate) treatment of cells or competition with heparin, heparan sulfate, and their oversulfated derivatives caused a marked reduction in pORF2 binding to the cells. Syndecan-1 is the most abundant proteoglycan present on these cells and, hence, plays a key role in pORF2 binding. Specificity is likely to be dictated by well-defined sulfation patterns on syndecans. We show that pORF2 binds syndecans predominantly via 6-O sulfation, indicating that binding is not entirely due to random electrostatic interactions. Using an in vitro infection system, we also showed a marked reduction in HEV infection of heparinase-treated cells. Our results indicate that, analogous to some enveloped viruses, a nonenveloped virus like HEV may have also evolved to use HSPGs as cellular attachment receptors.

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Partial Primary Structure of the 48- and 90-Kilodalton Core Proteins oF Cell Surface-Associated Heparan Sulfate Proteoglycans of Lung Fibroblasts

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)83534-7 ◽

1989 ◽

Vol 264 (12) ◽

pp. 7017-7024 ◽

Cited By ~ 6

Author(s):

P Marynen ◽

J Zhang ◽

J J Cassiman ◽

H Van den Berghe ◽

G David

Keyword(s):

Cell Surface ◽

Heparan Sulfate ◽

Primary Structure ◽

Heparan Sulfate Proteoglycans ◽

Lung Fibroblasts ◽

Core Proteins

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A Heparan Sulfate-Binding Cell Penetrating Peptide for Tumor Targeting and Migration Inhibition

BioMed Research International ◽

10.1155/2015/237969 ◽

2015 ◽

Vol 2015 ◽

pp. 1-15 ◽

Cited By ~ 5

Author(s):

Chien-Jung Chen ◽

Kang-Chiao Tsai ◽

Ping-Hsueh Kuo ◽

Pei-Lin Chang ◽

Wen-Ching Wang ◽

...

Keyword(s):

Cell Surface ◽

Heparan Sulfate ◽

Biological Activities ◽

Cell Penetrating Peptides ◽

Binding Activity ◽

Cellular Effects ◽

Cell Penetrating ◽

And Migration

As heparan sulfate proteoglycans (HSPGs) are known as co-receptors to interact with numerous growth factors and then modulate downstream biological activities, overexpression of HS/HSPG on cell surface acts as an increasingly reliable prognostic factor in tumor progression. Cell penetrating peptides (CPPs) are short-chain peptides developed as functionalized vectors for delivery approaches of impermeable agents. On cell surface negatively charged HS provides the initial attachment of basic CPPs by electrostatic interaction, leading to multiple cellular effects. Here a functional peptide (CPPecp) has been identified from critical HS binding region in hRNase3, a unique RNase family member within vitroantitumor activity. In this study we analyze a set of HS-binding CPPs derived from natural proteins including CPPecp. In addition to cellular binding and internalization, CPPecpdemonstrated multiple functions including strong binding activity to tumor cell surface with higher HS expression, significant inhibitory effects on cancer cell migration, and suppression of angiogenesisin vitroandin vivo. Moreover, different from conventional highly basic CPPs, CPPecpfacilitated magnetic nanoparticle to selectively target tumor sitein vivo. Therefore, CPPecpcould engage its capacity to be developed as biomaterials for diagnostic imaging agent, therapeutic supplement, or functionalized vector for drug delivery.

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Mutations in Biosynthetic Enzymes for the Protein Linker Region of Chondroitin/Dermatan/Heparan Sulfate Cause Skeletal and Skin Dysplasias

BioMed Research International ◽

10.1155/2015/861752 ◽

2015 ◽

Vol 2015 ◽

pp. 1-7 ◽

Cited By ~ 14

Author(s):

Shuji Mizumoto ◽

Shuhei Yamada ◽

Kazuyuki Sugahara

Keyword(s):

Heparan Sulfate ◽

Genetic Disorders ◽

Genetic Diseases ◽

Ehlers Danlos Syndrome ◽

Linker Region ◽

Desbuquois Dysplasia ◽

Core Proteins ◽

Wide Range ◽

Genes Encoding ◽

The Common

Glycosaminoglycans, including chondroitin, dermatan, and heparan sulfate, have various roles in a wide range of biological events such as cell signaling, cell proliferation, tissue morphogenesis, and interactions with various growth factors. Their polysaccharides covalently attach to the serine residues on specific core proteins through the common linker region tetrasaccharide, -xylose-galactose-galactose-glucuronic acid, which is produced through the stepwise addition of respective monosaccharides by four distinct glycosyltransferases. Mutations in the human genes encoding the glycosyltransferases responsible for the biosynthesis of the linker region tetrasaccharide cause a number of genetic disorders, called glycosaminoglycan linkeropathies, including Desbuquois dysplasia type 2, spondyloepimetaphyseal dysplasia, Ehlers-Danlos syndrome, and Larsen syndrome. This review focused on recent studies on genetic diseases caused by defects in the biosynthesis of the common linker region tetrasaccharide.

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