Absence Of Heparan Sulfate Proteoglycans Research Articles

Absence of heparan sulfate proteoglycans in Lewy bodies and Lewy neurites in Parkinson's disease brains

alpha-Synuclein is the major constituent of Lewy bodies and Lewy neurites in Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Relatively little is known about the exact mechanism of alpha-synuclein deposition and fibrillization in these alpha-synucleinopathies. In order to better understand the pathogenesis of alpha-synucleinopathies it is important to identify molecules that regulate the fibrillization of alpha-synuclein. Since it has been demonstrated that heparan sulfate proteoglycans (HSPGs) and glycosaminoglycans (GAGs) promote the conversion of non-fibrillar amyloid beta-protein (Abeta) into neurotoxic fibrillar Abeta in Alzheimer's disease, they might also be involved in alpha-synuclein aggregation. It was the aim of our study to examine the distribution pattern of these macromolecules in PD brains and the possible association with Lewy bodies and Lewy neurites. Although HSPGs clearly colocalized with senile plaques, we were unable to identify HSPGs or GAGs in Lewy bodies and Lewy neurites and therefore concluded that it is likely that alpha-synuclein fibrillization and stabilization occurs independently of the presence of HSPGs or GAGs.

RGD inclusion in VP3 provides adeno-associated virus type 2 (AAV2)-based vectors with a heparan sulfate-independent cell entry mechanism

Recombinant adeno-associated virus (AAV) has become an attractive vector system for a number of gene therapy paradigms. However, the utility of AAV vectors is often limited by the absence of heparan sulfate proteoglycan (HSPG), the virus's primary attachment receptor, on the desired target cell population. In order to achieve HSPG-independent gene delivery, several groups have shown that the endogenous tropism of AAV can be expand by genetically altering the viral capsid. However, the parameters of this developing technology have yet to be defined and it has not yet been determined if these modified vectors actually infect cells via these engineered interactions. Previously we constructed a series of insertion mutants spanning the AAV capsid protein gene and identified specific sites that can tolerate the insertion of small exogenous peptides. Here we describe a number of sites within the AAV capsid gene that can be used for the insertion of integrin-targeting peptide epitopes. Incorporation of an Arg-Gly-Asp (RGD)-containing peptide at these sites enables AAV to infect integrin-expressing cells independent of HSPG. Mutant AAV vectors displaying these peptide ligands can be produced to wild-type titer and have been shown to specifically interact with the targeted integrin receptors and mediate infection via this interaction. We report significant increases in gene transfer to Raji, K562, and SKOV-3 cell lines that express integrin, but little HSPG, suggesting that rAAV vectors displaying RGD peptides may be of great utility for treatment of neoplasms characterized by the deficiency of HSPG expression. We have also demonstrated that due to their expanded tropism, these novel vectors are capable of efficient transduction of AAV2-resistant tumors in vivo suggesting that they may offer significant therapeutic advantages.

Basement membrane of mouse bone marrow sinusoids shows distinctive structure and proteoglycan composition: a high resolution ultrastructural study.

Venous sinusoids in bone marrow are the site of a large-scale traffic of cells between the extravascular hemopoietic compartment and the blood stream. The wall of the sinusoids consists solely of a basement membrane interposed between a layer of endothelial cells and an incomplete covering of adventitial cells. To examine its possible structural specialization, the basement membrane of bone marrow sinusoids has now been examined by high resolution electron microscopy of perfusion-fixed mouse bone marrow. The basement membrane layer was discontinuous, consisting of irregular masses of amorphous material within a uniform 60-nm-wide space between apposing endothelial cells and adventitial cell processes. At maximal magnifications, the material was resolved as a random arrangement of components lacking the "cord network" formation seen in basement membranes elsewhere. Individual components exhibited distinctive ultrastructural features whose molecular identity has previously been established. By these morphological criteria, the basement membrane contained unusually abundant chondroitin sulfate proteoglycan (CSPG) revealed by 3-nm-wide "double tracks," and moderate amounts of both laminin as dense irregular coils and type IV collagen as 1-1.5-nm-wide filaments, together with less conspicuous amounts of amyloid P forming pentagonal frames. In contrast, 4.5-5-nm-wide "double tracks" characteristic of heparan sulfate proteoglycan (HSPG) were absent. The findings demonstrate that, in comparison with "typical" basement membranes in other tissues, the bone marrow sinusoidal basement membrane is uniquely specialized in several respects. Its discontinuous nature, lack of network organization, and absence of HSPG, a molecule that normally helps to maintain membrane integrity, may facilitate disassembly and reassembly of basement membrane material in concert with movements of adventitial cell processes as maturing hemopoietic cells pass through the sinusoidal wall: the exceptionally large quantity of CSPG may represent a reservoir of CD44 receptor for use in hemopoiesis.

Mechanisms of fibroblast growth factor 2 intracellular processing: a kinetic analysis of the role of heparan sulfate proteoglycans.

The interaction of fibroblast growth factor 2 (FGF-2) with heparan sulfate proteoglycans (HSPG) has been demonstrated to enhance receptor binding and alter the intracellular distribution of internalized FGF-2. In the present study, the intracellular fate of FGF-2 was analyzed in vascular smooth muscle cells (VSMC) under native and HSPG-deficient conditions. HSPG-deficient cells were generated by treatment with sodium chlorate. Cells were incubated with FGF-2 at 37 degrees C for prolonged periods (0-48 h) to allow for FGF-2 uptake and processing. Processing of FGF-2 occurred in stages. Initially a family of low molecular weight (LMW) fragments (4-10 kDa) were detected that accumulated to much higher ( approximately 10-fold) levels in native compared to heparan sulfate-deficient cells. Pulse-chase experiments revealed that the half-life of these LMW intermediates was significantly greater in native ( approximately 18 h) compared to HSPG-deficient cells ( approximately 4 h). Rate constants for FGF-2 processing were derived by modeling the uptake and processing of FGF-2 as a set of first-order differential equations. The kinetic analysis indicated that the greatest differences between native and HSPG-deficient VSMC was in the formation of LMW and further suggested that these FGF-2 products appear to represent a stable subpool of internal FGF-2 that is favored in cells that contain HSPG. Thus, HSPG might function as a cellular switch between immediate and prolonged signal activation by heparin-binding growth factors such as FGF-2. In the absence of HSPG, FGF-2 can interact with and activate its receptor, yet in the presence of HSPG, FGF-2 might be able to mediate prolonged or unique biological responses through intracellular processes.

Receptor-binding properties of a soluble form of human cytomegalovirus glycoprotein B.

The human cytomegalovirus (HCMV) glycoprotein B (gB) (also known as gpUL55) homolog is an important mediator of virus entry and cell-to-cell dissemination of infection. To examine the potential ligand-binding properties of gB, a soluble form of gB (gB-S) was radiolabeled, purified, and tested in cell-binding experiments. Binding of gB-S to human fibroblast cells was found to occur in a dose-dependent, saturable, and specific manner. Scatchard analysis demonstrated a biphasic plot with the following estimated dissociation constants (Kd): Kd1, 4.96 x 10(-6) M; Kd2, 3.07 x 10(-7) M. Cell surface heparan sulfate proteoglycans (HSPGs) were determined to serve as one class of receptors able to facilitate gB-S binding. Both HSPG-deficient Chinese hamster ovary (CHO) cells and fibroblast cells with enzymatically removed HSPGs had 40% reductions in gB-S binding, whereas removal of chondroitin sulfate had no effect. However, a significant proportion of gB-S was able to associate with the cell surface in the absence of HSPGs via an undefined nonheparin component. Binding affinity analysis of gB-S binding to wild-type CHO-K1 cells demonstrated biphasic binding kinetics (Kd1, 9.85 x 10(-6) M; Kd2, 4.03 x 10(-8) M), whereas gB-S binding to HSPG-deficient CHO-677 cells exhibited single-component binding kinetics (Kd, 7.46 x 10(-6) M). Together, these data suggest that gB-S associates with two classes of cellular receptors. The interaction of gB with its receptors is physiologically relevant, as evidenced by an inhibitory effect on HCMV entry when cells were pretreated with purified gB-S. This inhibition was determined to be manifested at the level of virus attachment. We conclude that gB is a ligand for HCMV that mediates an interaction with a cellular receptor(s) during HCMV infection.

Basic Fibroblast Growth Factor Binds Its Receptors, Is Internalized, and Stimulates DNA Synthesis in Balb/c3T3 Cells in the Absence of Heparan Sulfate

We have investigated the interaction of basic fibroblast growth factor (bFGF) with its receptors and heparan sulfate proteoglycans (HSPG). It has been suggested that in the absence of HSPG, cells are not able to bind bFGF or respond to treatment with bFGF. In our studies, Balb/c3T3 fibroblasts were treated with 50 mM sodium chlorate to completely inhibit (99%) sulfation of proteoglycans. We found that bFGF was able to bind, be internalized, and stimulate DNA synthesis in the absence of HSPG in a dose-dependent manner. bFGF bound to its receptors on chlorate-treated cells with a lower apparent affinity and no change in receptor number. To determine if this decreased affinity bFGF-receptor interaction is functional, we quantitatively analyzed bFGF internalization and stimulation of DNA synthesis in control and chlorate-treated cells. Endocytotic rate constants (ke) for chlorate-treated and control cells were ke = 0. 078 +/- 0.022 min-1 and ke = 0.043 +/- 0.012 min-1, respectively, suggesting that the process of bFGF internalization is not dramatically altered by HSPG. bFGF stimulated DNA synthesis to the same maximal level under both conditions, but chlorate-treated cells were significantly less responsive at low bFGF doses (approximately 10-fold increase in ED50). The differences observed for control and chlorate-treated cells in the dose-response curves for stimulation of DNA synthesis and receptor binding correlated directly, suggesting that receptors are equally capable of eliciting a mitogenic signal under both conditions. It is unlikely that these results are due to residual HSPG since heparinase (I and III) digestion of chlorate-treated cells had little effect. Although the presence of HSPG on the cell surface increases the affinity of bFGF for its receptors, our observations suggest that HSPG are not "absolutely" required for binding, internalization, or stimulation of mitogenic activity.

Enhanced binding and uptake of remnant lipoproteins by hepatic lipase-secreting hepatoma cells in culture.

Rat hepatoma McA-RH7777 cells transfected with a human hepatic lipase (HL) cDNA synthesized and secreted 50-80 ng of human HL/mg of cell protein at 4 h, approximately 50% of which was bound to cell-surface heparan sulfate proteoglycans (HSPG). The newly synthesized HL possessed enzymatic activity. When rabbit beta-very low density lipoproteins (beta-VLDL) and canine chylomicrons or chylomicron remnants were incubated with HL-secreting cells, remnant binding and uptake were enhanced 3-fold compared with nontransfected cells. Furthermore, fluorescence microscopy showed enhanced uptake of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-labeled beta-VLDL by the HL-transfected cells. When 125I-beta-VLDL were added to conditioned medium from HL-secreting cells, the HL in the media enhanced the binding and uptake of the remnant lipoproteins by nontransfected cells about 3-fold. Likewise, surface-bound HL (without HL in the medium) also was able to mediate the enhanced binding of the remnants. This HL-enhanced binding was shown to be mediated by an interaction with cell-surface HSPG. Heparinase treatment to remove cell-surface HSPG or chlorate treatment to prevent HSPG sulfation of the HL-secreting cells abolished all the HL-mediated enhanced binding and uptake. Furthermore, heparinase pretreatment of nontransfected cells prevented the enhanced binding and uptake of beta-VLDL incubated with conditioned medium from HL-secreting cells. As binding was not enhanced in the absence of HSPG, an HL-HSPG initial interaction appears essential. Addition of apolipoprotein (apo) E to the beta-VLDL did not facilitate HL-mediated binding and uptake; in fact, beta-VLDL from apoE-null mice demonstrated a similar degree of enhanced binding as did rabbit beta-VLDL with or without added apoE. On the other hand, beta-VLDL from transgenic mice overexpressing binding-defective apoE(Arg142-->Cys) did not display any enhanced binding and uptake by the HL-secreting cells, and it appears that the apoE(Arg142-->Cys) actually inhibited the HL-mediated interaction. This mutant form of apoE is associated with a dominant mode of expression of type III hyperlipoproteinemia in contrast to the more commonly occurring recessive disorder. Impaired HL interaction with the apoE(Arg142-->Cys) beta-VLDL may contribute to remnant lipoprotein accumulation in the plasma of patients with this mutant form of apoE. Thus, HL contributes to the enhanced cell association of specific types of remnant lipoproteins by initiating their binding to cell-surface HSPG.

Characterization of an inhibitor of neuronal plasminogen activator released by heart cells

A basic understanding of growth cone dynamics and developmental events involving growth cones requires an understanding of the function and regulation of molecules associated with and released by growth cones. Rat sympathetic neurons in culture release a urokinase-like plasminogen activator from their distal processes and/or growth cones (Pittman, 1985a). When sympathetic neurons are grown in cocultures with heart cells, however, plasminogen activator activity is not detected. The absence of plasminogen activator activity in cocultures of sympathetic neurons and heart cells appears to be due to the release of an inhibitor of plasminogen activator by heart cells. This inhibitor has a molecular weight of approximately 50 kDa in the presence of SDS and apparent molecular weights of approximately 50 and greater than 2000 kDa under native conditions. A significant fraction of the large-molecular-weight form of the inhibitor is converted to the smaller form following treatment with heparinase. Extremely stable complexes of 68 and 80 kDa are formed between the heart inhibitor and the plasminogen activator, urokinase, such that the complexes withstand boiling in SDS/mercaptoethanol. The data are consistent with the formation of an 80 kDa urokinase-inhibitor complex in the presence of heparan sulfate proteoglycan and a 68 kDa complex in the absence of heparan sulfate proteoglycan. A highly purified preparation of the heart inhibitor produces a 2- to 3-fold increase in neurite outgrowth from sympathetic neurons. These data indicate that the activity of the plasminogen activator released by sympathetic neurons can be regulated by a normal target tissue and that this regulation may result in increased neurite outgrowth from the neurons.

Lack of heparan sulfate proteoglycan in a discontinuous and irregular placental basement membrane

A discontinuous basement membrane of variable width that surrounds spongiotrophoblast cells of rat placenta was examined for the presence of type IV collagen, laminin, a heparan sulfate proteoglycan, entactin, and fibronectin using monospecific antibodies or antisera and the indirect peroxidase technique. At the level of the light microscope, the basement membrane was immunostained for type IV collagen, laminin, entactin, and fibronectin. Heparan sulfate proteoglycan immunostaining, however, was virtually absent even after pretreatment of sections with 0.1 N acetic acid, pepsin (0.1 μg/ml) or 0.13 M sodium borohydride. Examination in the electron microscope confirmed the lack of immunostaining for heparan sulfate proteoglycan, whereas the other substances were mainly localized to the lamina densa part of the basement membrane. The absence of heparan sulfate proteoglycan in this discontinuous and irregular basement membrane even though type IV collagen, laminin, entactin, and fibronectin are present, suggests that heparan sulfate proteoglycan may have a structural role in the formation of basement membrane.