Insight into the role of HSPG in the cellular uptake of apolipoprotein E-derived peptide micelles and liposomes

Liposomes and micellar carriers equipped with targeting and cellular uptake mediating peptides have attracted attention for numerous applications. The optimization of the carrier requires an understanding of how their properties influence target cell recognition and uptake. We developed a dipalmitoylated apolipoprotein E-derived peptide, named P2A2 as promising vector to mediate cellular uptake of potential micellar and liposomal carriers. Confocal laser scanning microscopy (CLSM) and fluorescence-activated cell sorting (FACS) were used to get insight into the internalization mediated by carboxyfluoresceine-labeled P2fA2 and the all-D amino acid analogue P2fa2 into brain capillary endothelial cells. Both peptide micelles and liposomes entered cells via endocytosis. Cell surface heparan sulfate proteoglycans (HSPGs) were involved in the internalization process of peptide-bearing liposomes characterized by a diameter of 100 nm, a low surface density of 100 peptide molecules per vesicle and a helical conformation of the vector. In contrast, peptide micelles characterized by a diameter of about 10 nm, a high peptide density caused by 19 associated molecules and a high conformational flexibility of the vector sequence did not address HSPG. Unspecific interactions between the carriers and membrane constituents predominate the two uptake processes but stereospecific components seem to be involved. Both routes differ with respect to transport efficiency. The results provide a prospective basis to optimize liposomes and micelles as drug delivery systems.     

Basic fibroblast growth factor-syndecan complex at cell surface or immobilized to matrix promotes cell growth.

Promotion of cell growth and differentiation by growth factors during early development and organ formation are both temporally and spatially very precise. Syndecan is a well characterized integral membrane proteoglycan that binds several extracellular matrix components via its heparan sulfate chains and is therefore suggested to participate in cell regulation. Syndecan-like molecules, as low affinity receptors for heparin-binding growth factors, have been recently suggested to also regulate growth factor activity. Heparin/heparan sulfate interaction is required before, e.g. basic fibroblast growth factor (bFGF) can associate with its high affinity cell surface receptors and trigger signal transduction. In this paper we show that syndecan, but not free heparan sulfate chains, can simultaneously bind both bFGF and extracellular matrix molecules. Moreover, increased DNA synthesis of 3T3 cells was observed when the 3T3 cells were exposed to beads coated with the fibronectin-syndecan-bFGF complex, indicating that bFGF remains biologically active even when immobilized to matrix via the heparan sulfate chains of syndecan. Finally, when bFGF was bound to the surface of another cell type (epithelial), co-culture with 3T3 cells stimulated 3T3 cell growth. Therefore, we suggest that syndecan-like molecules may determine sites of growth factor action at cell-matrix and cell-cell interfaces.     

Modulations of glypican-1 heparan sulfate structure by inhibition of endogenous polyamine synthesis. Mapping of spermine-binding sites and heparanase, heparin lyase, and nitric oxide/nitrite cleavage sites

Cell surface heparan sulfate proteoglycans facilitate uptake of growth-promoting polyamines (Belting, M., Persson, S., and Fransson, L.-A. (1999) Biochem. J. 338, 317-323; Belting, M., Borsig, L., Fuster, M. M., Brown, J. R., Persson, L., Fransson, L.-A., and Esko, J. D. (2001) Proc. Natl. Acad. Sci. U. S. A., in press). Here, we have analyzed the effect of polyamine deprivation on the structure and polyamine affinity of the heparan sulfate chains in various glypican-1 glycoforms synthesized by a transformed cell line (ECV 304). Heparan sulfate chains of glypican-1 were either cleaved with heparanase at sites embracing the highly modified regions or with nitrite at N-unsubstituted glucosamine residues. The products were separated and further degraded by heparin lyase to identify sulfated iduronic acid. Polyamine affinity was assessed by chromatography on agarose substituted with the polyamine spermine. In heparan sulfate made by cells with undisturbed endogenous polyamine synthesis, free amino groups were restricted to the unmodified, unsulfated segments, especially near the core protein. Spermine high affinity binding sites were located to the modified and highly sulfated segments that were released by heparanase. In cells with up-regulated polyamine uptake, heparan sulfate contained an increased number of clustered N-unsubstituted glucosamines and sulfated iduronic acid residues. This resulted in a greater number of NO/nitrite-sensitive cleavage sites near the potential spermine-binding sites. Endogenous degradation by heparanase and NO-derived nitrite in polyamine-deprived cells generated a separate pool of heparan sulfate oligosaccharides with an exceptionally high affinity for spermine. Spermine uptake in polyamine-deprived cells was reduced when NO/nitrite-generated degradation of heparan sulfate was inhibited. The results suggest a functional interplay between glypican recycling, NO/nitrite-generated heparan sulfate degradation, and polyamine uptake.     

Bovine arterial smooth muscle cells synthesize two functionally different proteoheparan sulfate species

Cultured arterial smooth muscle cells synthesize two proteoheparan sulfate species. One is found associated with the cells, whereas the other is excreted into the medium. The two proteoheparan sulfates have similar hydrodynamic sizes but differ in the Mr of their core proteins. The cell-associated proteoheparan sulfate has a Mr of 92,000 while that of soluble proteoheparan sulfate is 38,000. The cell-associated and the soluble proteoheparan sulfate species differ in their ability to suppress the proliferation of smooth muscle cells. When added to the culture medium 2-5 micrograms/ml of the cell-associated and 20-25 micrograms/ml of the soluble proteoheparan sulfate species inhibit the growth of smooth muscle cells half maximally. The antiproliferative potency of both species resides in the heparan sulfate chains. Commercially available heparin has no antiproliferative effect and is not able to prevent the antiproliferative action of cellular heparan sulfate. In contrast to heparin, none of the heparan sulfate preparations has anticoagulant activity. Smooth muscle cells endocytose the soluble heparan sulfate at a rate three to four times higher than that of the cell-associated heparan sulfate. The data suggest that the cell-associated and the soluble proteoheparan sulfate species are separate and possibly genetically distinct molecules. Furthermore, the structural determinants for antiproliferative activity and the recognition sites for endocytotic uptake appear to be different.     

Fibronectin on the Surface of Myeloma Cell-derived Exosomes Mediates Exosome-Cell Interactions

Exosomes regulate cell behavior by binding to and delivering their cargo to target cells; however, the mechanisms mediating exosome-cell interactions are poorly understood. Heparan sulfates on target cell surfaces can act as receptors for exosome uptake, but the ligand for heparan sulfate on exosomes has not been identified. Using exosomes isolated from myeloma cell lines and from myeloma patients, we identify exosomal fibronectin as a key heparan sulfate-binding ligand and mediator of exosome-cell interactions. We discovered that heparan sulfate plays a dual role in exosome-cell interaction; heparan sulfate on exosomes captures fibronectin, and on target cells it acts as a receptor for fibronectin. Removal of heparan sulfate from the exosome surface releases fibronectin and dramatically inhibits exosome-target cell interaction. Antibody specific for the Hep-II heparin-binding domain of fibronectin blocks exosome interaction with tumor cells or with marrow stromal cells. Regarding exosome function, fibronectin-mediated binding of exosomes to myeloma cells activated p38 and pERK signaling and expression of downstream target genes DKK1 and MMP-9, two molecules that promote myeloma progression. Antibody against fibronectin inhibited the ability of myeloma-derived exosomes to stimulate endothelial cell invasion. Heparin or heparin mimetics including Roneparstat, a modified heparin in phase I trials in myeloma patients, significantly inhibited exosome-cell interactions. These studies provide the first evidence that fibronectin binding to heparan sulfate mediates exosome-cell interactions, revealing a fundamental mechanism important for exosome-mediated cross-talk within tumor microenvironments. Moreover, these results imply that therapeutic disruption of fibronectin-heparan sulfate interactions will negatively impact myeloma tumor growth and progression.     

Distinct effects on heparan sulfate structure by different active site mutations in NDST-1

Heparan sulfate polymerization and modification take place in the Golgi compartment. The modification reactions are initiated by glucosaminyl N-deacetylase/N-sulfotransferase (NDST), a bifunctional enzyme that removes N-acetyl groups from selected N-acetyl-d-glucosamine units followed by N-sulfation of the generated free amino groups. Four isoforms of NDST have been identified. NDST-1 and -2 have a wide and largely overlapping tissue distribution, but it is not known if they can act on the same heparan sulfate chain. We have introduced point mutations into NDST-1 cDNA, which selectively destroy the N-deacetylase or N-sulfotransferase activity of the enzyme [Wei, Z., and Swiedler, S. J. (1999) J. Biol. Chem. 274, 1966-70 and Sueyoshi, T., et al. (1998) FEBS Lett. 433, 211-4]. Stable 293 cell lines expressing the NDST-1 mutants were then generated. Structural analyses of heparan sulfate synthesized by these cells and by cells overexpressing wild-type NDST-1 demonstrate that the N-deacetylation step is not only prerequisite but also rate-limiting, determining the degree of N-sulfation. Transfection of mutant NDST-1 lacking N-deacetylase activity had no effect on heparan sulfate sulfation, while cells expressing wild-type enzyme or NDST-1 lacking N-sulfotransferase activity both resulted in the production of oversulfated heparan sulfate. Since no increase in the amount of N-unsubstituted glucosamine residues was seen after transfection of the mutant lacking N-sulfotransferase activity, the results also suggest that two different enzyme molecules can act on the same glucosamine unit. In addition, we show that oversulfation of heparan sulfate produced by cells tranfected with wild-type NDST-1 or the mutant lacking N-sulfotranferase activity results in decreased sulfation of chondroitin sulfate.     

Heparin releasable and nonreleasable forms of heparan sulfate proteoglycan are found on the surfaces of cultured porcine aortic endothelial cells

Evidence suggests that endothelial cell layer heparan sulfate proteoglycans include a variety of different sized molecules which most likely contain different protein cores. In the present report, approximately half of endothelial cell surface associated heparan sulfate proteoglycan is shown to be releasable with soluble heparin. The remaining cell surface heparan sulfate proteoglycan, as well as extracellular matrix heparan sulfate proteoglycan, cannot be removed from the cells with heparin. The heparin nonreleasable cell surface proteoglycan can be released by membrane disrupting agents and is able to intercalate into liposomes. When the heparin releasable and nonreleasable cell surface heparan sulfate proteoglycans are compared, differences in proteoglycan size are also evident. Furthermore, the intact heparin releasable heparan sulfate proteoglycan is closer in size to proteoglycans isolated from the extracellular matrix and from growth medium than to that which is heparin nonreleasable. These data indicate that cultured porcine aortic endothelial cells contain at least two distinct types of cell surface heparan sulfate proteoglycans, one of which appears to be associated with the cells through its glycosaminoglycan chains. The other (which is more tightly associated) is probably linked via a membrane intercalated protein core.Abbreviations ECM extracellular matrix - HSPG heparan sulfate proteoglycan - PAE porcine aortic endothelial - PBS phosphate buffered saline     

Heparin and heparan sulfate binding sites on B-16 melanoma cells

We have reported previously that the production of a tumor cell factor that stimulates synthesis of fibroblast collagenase is influenced by a fibroblast-deposited matrix component, possibly heparan sulfate-proteoglycan. In this study, binding sites for heparin and heparan sulfate on mouse B-16 melanoma cells have been demonstrated. Binding of 3H-heparin and 35S-heparan sulfate has been shown to occur to whole cells, isolated membranes, and to a component(s) of detergent extracts of the membranes. Scatchard analysis of binding of 3H-heparin yielded a Kd of 2-5 x 10(-8) M and a Bmax of 0.5 x 10(7) heparin molecules bound per cell. Binding of 35S-heparan sulfate was of at least an order of magnitude lower affinity than heparin, but the Bmax was similar to that for heparin. Competition studies showed that 35S-heparan sulfate binding was inhibited totally by heparin and heparan sulfate and partially by dermatan sulfate, but no inhibition was obtained with hyaluronate or chondroitin sulfate. Binding of 3H-heparin was inhibited totally by heparin but to different extents by preparations of heparan sulfate from different tissue sources. The heparin/heparan sulfate binding activity is a protein(s) because it is destroyed by treatment with trypsin. Binding of 3H-heparin to transblots of the detergent extract of the B-16 cell membranes indicated that at least part of the binding activity is a 14,000-dalton protein.     

Endothelial cell-derived heparan sulfate binds basic fibroblast growth factor and protects it from proteolytic degradation

Cultured bovine capillary endothelial (BCE) cells were found to synthesize and secrete high molecular mass heparan sulfate proteoglycans and glycosaminoglycans, which bound basic fibroblast growth factor (bFGF). The secreted heparan sulfate molecules were purified by DEAE cellulose chromatography, followed by Sepharose 4B chromatography and affinity chromatography on immobilized bFGF. Most of the heparinase-sensitive sulfated molecules secreted into the medium by BCE cells bound to immobilized bFGF at low salt concentrations. However, elution from bFGF with increasing salt concentrations demonstrated varying affinities for bFGF among the secreted heparan sulfate molecules, with part of the heparan sulfate requiring NaCl concentrations between 1.0 and 1.5 M for elution. Cell extracts prepared from BCE cells also contained a bFGF-binding heparan sulfate proteoglycan, which could be released from the intact cells by a short proteinase treatment. The purified bFGF-binding heparan sulfate competed with 125I-bFGF for binding to low-affinity binding sites but not to high-affinity sites on the cells. Heparan sulfate did not interfere with bFGF stimulation of plasminogen activator activity in BCE cells in agreement with its lack of effect on binding of 125I-bFGF to high-affinity sites. Soluble bFGF was readily degraded by plasmin, whereas bFGF bound to heparan sulfate was protected from proteolytic degradation. Treatment of the heparan sulfate with heparinase before addition of plasmin abolished the protection and resulted in degradation of bFGF by the added proteinase. The results suggest that heparan sulfate released either directly by cells or through proteolytic degradation of their extracellular milieu may act as carrier for bFGF and facilitate the diffusion of locally produced growth factor by competing with its binding to surrounding matrix structures. Simultaneously, the secreted heparan sulfate glycosaminoglycans protect the growth factor from proteolytic degradation by extracellular proteinases, which are abundant at sites of neovascularization or cell invasion.     

Human recombinant interleukin-1 beta- and tumor necrosis factor alpha-mediated suppression of heparin-like compounds on cultured porcine aortic endothelial cells

Cytokines are known to tip the balance of the coagulant-anticoagulant molecules on the endothelial cell surface toward intravascular coagulation. Their effects on endothelial cell surface-associated heparin-like compounds have not been examined yet. Incorporation of [35S]sulfate into heparan sulfate on cultured porcine aortic endothelial cells was suppressed by human recombinant interleukin-1 beta (rIL-1 beta) or tumor necrosis factor alpha (rTNF alpha) in a dose- and time-dependent manner with little effect on cell number, protein content, and [3H]leucine incorporation of cells. Maximal inhibition was achieved by incubation of cells with 100 ng/ml of rIL-1 beta or 5 ng/ml of rTNF alpha for 12-24 hours, resulting in a reduction of the synthesis of heparan sulfate on the cell surface by approximately 50%. The dose dependency was consistent with that seen in the stimulation of endothelial cell procoagulant activity by each cytokine. The suppression of heparan sulfate synthesis was sustained for at least 48 hours after pretreatment of cells with cytokines and was unchanged after the addition of indomethacin or polymyxin B. The rate of degradation of prelabeled 35S-heparan sulfate on the cell surface was not altered by cytokine treatments. Neither the size, the net negative charge, nor the proportion of the molecule with high affinity for antithrombin III of endothelial cell heparan sulfate was changed by cytokines. Furthermore, specific binding of 125I-labeled antithrombin III to the endothelial cell surface was reduced to 40-60% of control by cytokines. In parallel with reduction in binding, antithrombin III cofactor (heparin-like) activity was partially diminished in cytokine-treated endothelial cells. Thus, cytokine-mediated suppression of heparin-like substance on endothelial cells appears to be another cytokine-inducible endothelial effects affecting coagulation.     

Pathway for polyarginine entry into mammalian cells

Cationic peptides known as protein transduction domains (PTDs) provide a means to deliver molecules into mammalian cells. Here, nonaarginine (R(9)), the most efficacious of known PTDs, is used to elucidate the pathway for PTD internalization. Although R(9) is found in the cytosol as well as the nucleolus when cells are fixed, this peptide is observed only in the endocytic vesicles of live cells. Colocalization studies with vesicular markers confirm that PTDs are internalized by endocytosis rather than by crossing the plasma membrane. The inability of R(9) to enter living cells deficient in heparan sulfate (HS) suggests that binding to HS is necessary for PTD internalization. This finding is consistent with the high affinity of R(9) for heparin (K(d) = 109 nM). Finally, R(9) is shown to promote the leakage of liposomes but only at high peptide:lipid ratios. These and other data indicate that the PTD-mediated delivery of molecules into live mammalian cells involves (1) binding to cell surface HS, (2) uptake by endocytosis, (3) release upon HS degradation, and (4) leakage from endocytic vesicles.     

Crotamine mediates gene delivery into cells through the binding to heparan sulfate proteoglycans

Recently we have shown that crotamine, a toxin from the South American rattlesnake Crotalus durissus terrificus venom, belongs to the family of cell-penetrating peptides. Moreover, crotamine was demonstrated to be a marker of centrioles, of cell cycle, and of actively proliferating cells. Herein we show that this toxin at non-toxic concentrations is also capable of binding electrostatically to plasmid DNA forming DNA-peptide complexes whose stabilities overcome the need for chemical conjugation for carrying nucleic acids into cells. Interestingly, crotamine demonstrates cell specificity and targeted delivery of plasmid DNA into actively proliferating cells both in vitro and in vivo, which distinguishes crotamine from other known natural cell-penetrating peptides. The mechanism of crotamine penetration and cargo delivery into cells was also investigated, showing the involvement of heparan sulfate proteoglycans in the uptake phase, which is followed by endocytosis and peptide accumulation within the acidic endosomal vesicles. Finally, the permeabilization of endosomal membranes induced by crotamine results in the leakage of the vesicles contents to the cell cytosol.     

Glycosaminoglycan Composition of PC 12 Pheochromocytoma Cells: A Comparison with PC12D Cells, a New Subline of PC12 Cells

PC12D cells, a new subline of conventional PC12 cells, respond not only to nerve growth factor but also to cyclic AMP by extending their neurites. These cells are flat in shape and are similar in appearance to PC12 cells that have been treated with nerve growth factor for a few days. In both cell lines, we have characterized the glycosaminoglycans, the polysaccharide moieties of proteoglycans, which are believed to play an important role in cell adhesion and in cell morphology. Under the present culture conditions, only chondroitin sulfate was detected in the media from PC12 and PC12D cells, whereas both chondroitin sulfate and heparan sulfate were found in the cell layers. The levels of cell-associated heparan sulfate and chondroitin sulfate were about twofold and fourfold higher in PC12D cells than in PC12 cells, respectively. Compared to PC12 cells, the amounts of [35S]sulfate incorporated for 48 h into chondroitin sulfate were twofold lower but those into heparan sulfate were 35% higher in PC12D cells. The amount of chondroitin sulfate released by PC12D cells into the medium was about a half of that released by PC12 cells. The ratio of [35S]sulfate-labeled heparan sulfate to chondroitin sulfate was 6.2 in PC12D cells and 2.2 in PC12 cells. These results suggest that there may be some correlation between the increase in content of glycosaminoglycans and the change in cell morphology, which is followed by neurite outgrowth.     

A catalytically independent physiological function for human acute phase protein group IIA phospholipase A2: cellular uptake facilitates cell debris removal

Human group IIA phospholipase A2 (IIA PLA2) is an acute phase protein first identified at high concentrations in synovial fluid from patients with rheumatoid arthritis. Its physiological role has since been debated; the enzyme has a very high affinity for anionic phospholipid interfaces but expresses almost zero activity with zwitterionic phospholipid substrates, because of a lack of interfacial binding. We have prepared the cysteine-containing mutant (S74C) to allow the covalent attachment of fluorescent reporter groups. We show that fluorescently labeled IIA was taken up by phorbol 12-myristate 13-acetate-activated THP-1 cells in an energy-dependent process involving cell surface heparan sulfate proteoglycans. Uptake concurrently involved significant cell swelling, characteristic of macropinocytosis and the fluorescent enzyme localized to the nucleus. The endocytic process did not necessitate enzyme catalysis, ruling out membrane phospholipid hydrolysis as an essential requirement. The enzyme produced supramolecular aggregates with anionic phospholipid vesicles as a result of bridging between particles, a property that is unique to this globally cationic IIA PLA2. Uptake of such aggregates labeled with fluorescent anionic phospholipid was dramatically enhanced by the IIA protein, and uptake involved binding to heparan sulfate proteoglycans on activated THP-1 cells. A physiological role for this protein is proposed that involves the removal of anionic extracellular cell debris, including anionic microparticles generated as a result of trauma, infection, and the inflammatory response, and under such conditions serum levels of IIA PLA2 can increase approximately 1000-fold. A similar pathway may be significant in the uptake into cells of anionic vector DNA involving cationic lipid transfection protocols.     

Efficient infection of cells in culture by type O foot-and-mouth disease virus requires binding to cell surface heparan sulfate.

Foot-and-mouth disease virus (FMDV) enters cells by attaching to cellular receptor molecules of the integrin family, one of which has been identified as the RGD-binding integrin alpha(v)beta3. Here we report that, in addition to an integrin binding site, type O strains of FMDV share with natural ligands of alpha(v)beta3 (i.e., vitronectin and fibronectin) a specific affinity for heparin and that binding to the cellular form of this sulfated glycan, heparan sulfate, is required for efficient infection of cells in culture. Binding of the virus to paraformaldehyde-fixed cells was powerfully inhibited by agents such as heparin, that compete with heparan sulfate or by agents that compete for heparan sulfate (platelet factor 4) or that inactivate it (heparinase). Neither chondroitin sulfate, a structurally related component of the extracellular matrix, nor dextran sulfate appreciably inhibited binding. The functional importance of heparan sulfate binding was demonstrated by the facts that (i) infection of live cells by FMDV could also be blocked specifically by heparin, albeit at a much higher concentration of inhibitor; (ii) pretreatment of cells with heparinase reduced the number of plaques formed compared with that for untreated cells; and (iii) mutant cell lines deficient in heparan sulfate expression were unable to support plaque formation by FMDV, even though they remained equally susceptible to another picornavirus, bovine enterovirus. The results show that entry of type O FMDV into cells is a complex process and suggest that the initial contact with the cell surface is made through heparan sulfate.     

Syndecan from embryonic tooth mesenchyme binds tenascin.

Syndecan is a cell surface heparan sulfate-rich proteoglycan found on various epithelial cells but also in some embryonic mesenchymal tissues. We have immunoisolated syndecan from embryonic tooth mesenchyme that appeared as a 250-300-kDa molecule (Kav = 0.3 in Sepharose 4B), containing only heparan sulfate side chains (Mr = 35,000). Northern analysis of whole tooth germs and tooth mesenchymes also revealed high expression of syndecan mRNAs (2.6 and 3.4 kilobases). In the binding assay utilizing nitrocellulose as a solid phase to immobilize matrix molecules, syndecan immunoisolated from tooth mesenchyme revealed binding to tenascin, and this interaction was shown to be mediated via heparan sulfate side chains. In contrast, syndecan from mouse mammary epithelial cells showed only weak interaction with tenascin. We propose that syndecan and tenascin may represent interactions of a cell surface receptor and a matrix ligand involved in mesenchymal cell condensation and differentiation during early organogenesis.     

Disulfide-bonded aggregates of heparan sulfate proteoglycans

Heparan sulfate proteoglycans have been isolated from Swiss mouse 3T3 cells by using two nondegradative techniques: extraction with 4 M guanidine or 2.5% 1-butanol. These proteoglycans were separated from copurifying chondroitin sulfate proteoglycans by using ion-exchange chromatography on DEAE-cellulose in the presence of 2 M urea. The purified heparan sulfate proteoglycans are substantially smaller, ca. Mr 20 000, than those isolated from these same cells with trypsin, ca. Mr 720 000 [Johnston, L.S., Keller, K. L., & Keller, J. M. (1979) Biochim. Biophys. Acta 583, 81-94]. However, all of the heparan sulfate proteoglycans extracted by these three methods contain similar glycosaminoglycan chains (Mr 7500) and are derived from the same pool of cell surface associated molecules. The trypsin-released heparan sulfate proteoglycan (ca. Mr 720 000) can be significantly reduced in size (ca. Mr 33 000) under strong denaturing conditions in the presence of the disulfide reducing agent dithiothreitol, which suggests that this form of the molecule is a disulfide-bonded aggregate. The heparan sulfate proteoglycan isolated from the medium also undergoes a significant size reduction in the presence of dithiothreitol, indicating that a similar aggregate is formed as part of the normal release of heparan sulfate proteoglycans into the medium. These results suggest that well-shielded disulfide bonds between individual heparan sulfate proteoglycan monomers may account for the large variation in sizes which has been reported for heparan sulfate proteoglycans isolated from a variety of cells and tissues with a variety of extraction procedures.     

High molecular weight kininogen utilizes heparan sulfate proteoglycans for accumulation on endothelial cells

Kininogens, the high molecular weight precursor of vasoactive kinins, bind to a wide variety of cells in a specific, reversible, and saturable manner. The cell docking sites have been mapped to domains D3 and D5(H) of kininogens; however, the corresponding cellular acceptor sites are not fully established. To characterize the major cell binding sites for kininogens exposed by the endothelial cell line EA.hy926, we digested intact cells with trypsin and other proteases and found a time- and concentration-dependent loss of (125)I-labeled high molecular weight kininogen (H-kininogen) binding capacity (up to 82%), indicating that proteins are crucially involved in kininogen cell attachment. Cell surface digestion with heparinases similarly reduced kininogen binding capacity (up to 78%), and the combined action of heparinases and trypsin almost eliminated kininogen binding (up to 85%), suggesting that proteoglycans of the heparan sulfate type are intimately involved. Consistently, inhibitors such as p-nitrophenyl-beta-d-xylopyranoside and chlorate interfering with heparan sulfate proteoglycan biosynthesis reduced the total number of kininogen binding sites in a time- and concentration-dependent manner (up to 67%). In vitro binding studies demonstrated that biotinylated H-kininogen binds to heparan sulfate glycosaminoglycans via domains D3 and D5(H) and that the presence of Zn(2+) promotes this association. Cloning and over-expression of the major endothelial heparan sulfate-type proteoglycans syndecan-1, syndecan-2, syndecan-4, and glypican in HEK293t cells significantly increased total heparan sulfate at the cell surface and thus the number of kininogen binding sites (up to 3. 3-fold). This gain in kininogen binding capacity was completely abolished by treating transfected cells with heparinases. We conclude that heparan sulfate proteoglycans on the surface of endothelial cells provide a platform for the local accumulation of kininogens on the vascular lining. This accumulation may allow the circumscribed release of short-lived kinins from their precursor molecules in close proximity to their sites of action.