Affinity For Heparan Sulfate Proteoglycans Research Articles

Chemically modified galactans of Grateloupia indica: From production to in vitro antiviral activity

Herpes simplex viruses (HSVs) have an affinity for heparan sulfate proteoglycans on cell surfaces, which is a determinant for virus entry. Herein, several sulfated galactans that mimic the active domain of the entry receptor were employed to prevent HSV infection. They were produced from Grateloupia indica using chlorosulfonic acid-pyridine (ClSO3H.Py)/N,N-dimethylformamide reagent (fraction G-402), SO3.Py/DMF reagent (G-403), or by aqueous extraction (G-401). These galactans contained varied molecular masses (33–55 kDa), and sulfate contents (12–20 %), and have different antiviral activities. Especially, the galactan (G-402) generated by using ClSO3H.Py/DMF, a novel reagent, exhibited the highest level of antiviral activity (EC50 = 0.36 μg/mL) compared to G-403 (EC50 = 15.6 μg/mL) and G-401 (EC50 = 17.9 μg/mL). This most active sulfated galactan possessed a linear chain containing β-(1 → 3)- and α-(1 → 4)-linked Galp units with sulfate group at the O-2/4/6 and O-2/3/6 positions, respectively. The HSV-1 and HSV-2 strains were specifically inhibited by this novel 33 ± 15 kDa galactan, which also blocked the virus from entering the host cell. These results highlight the significant potential of this sulfated galactan for antiviral research and drug development. Additionally, the reagent used for the effective conversion of galactan hydroxy groups to sulfate during extraction may also be useful for the chemical transformation of other natural products.

Heparan sulfate proteoglycans-mediated targeted delivery of TGF-β1-binding peptide to liver for improved anti-liver fibrotic activity in vitro and in vivo

Elevated expressions of transforming growth factor β1 (TGF-β1) have been implicated in the pathogenesis of liver fibrosis, thus attenuating the excessive TGF-β1's activity by TGF-β1-binding peptide is an ideal strategy for the treatment of liver fibrosis. However, the application of small peptide as a pharmaceutical agent is obstacle due to difficult preparation and non-selective delivery. The I-plus sequences of circumsporozoite protein (CSP-I) possesses high affinity for heparan sulfate proteoglycans, which are primarily located on liver tissues. TGF-β1-binding peptide P15 holds specific ability of binding to TGF-β1. In this study, we describe an approach to efficiently preparing liver-targeting peptide P15-CSP-I, which is conjugation of the sequences of P15 to the N-terminus of CSP-I, from the cleavage of biological macromolecule SUMO-tagged P15-CSP-I. In vitro and ex vivo binding assay showed that P15-CSP-I specifically targeted to the hepatocytes and liver tissues. Moreover, P15-CSP-I inhibited cell proliferation, migration and invasion, and decreased fibrosis-related proteins expression in TGF-β1-activated HSCs in vitro. Furthermore, P15-CSP-I ameliorated liver morphology and decreased the fibrosis responses in vivo. Taken together, P15-CSP-I may be a potential candidate for targeting therapy on liver fibrosis due to its high efficient preparation, specific liver-targeting potential and improved anti-liver fibrotic activity.

Hedgehog Nanopackages Ready for Dispatch

Hedgehog proteins are intercellular long-range signaling molecules that spread within tissues and activate gene expression during development. Vyas et al. (2008) propose that Hedgehog forms nanometer-sized oligomers that localize in proteoglycan-rich clusters at the surface of cells expressing Hedgehog. This nanoscale organization and enrichment in clusters ensures that Hedgehog is able to spread and activate signaling over many cell diameters.

Growing and shaping the vascular tree: multiple roles for VEGF.

Vascular Endothelial Growth Factor (VEGF) is the most potent and ubiquitous vascular growth factor known to date. Yet, prior to its description as a secreted mitogen for endothelial cells, it was identified as a vascular permeability factor. These seemingly disparate avenues of discovery highlight VEGF's ability to control many distinct aspects of endothelial cell behaviour, including proliferation, migration, specialisation and survival. The versatility of VEGF as a patterning molecule is likely linked to its association with various signalling receptor complexes, but also its expression in several isoforms with a differential affinity for heparan sulfate proteoglycans in the extracellular matrix. In contrast to the absolute requirement for all known VEGF receptors, the presence of only a single VEGF isoform is sufficient for vascular development. However, the isoforms serve as exquisite tools for the fine patterning of growing vessel networks during embryogenesis and in postnatal life.

Pathogenesis of beta2-microglobulin amyloidosis.

Hemodialysis-related amyloidosis is a relatively new form of systemic amyloidosis, with beta2-microglobulin (B2M) being identified as the major constituent protein. Most of the clinical findings are related to amyloid deposition in osseo-articular tissues. B2M amyloid deposits first appear in the cervical intervertebral discs, which are well known to be susceptible to mechanical stress. A close relationship between changes of microenvironment caused by such stress and amyloid deposition is highly suggested. In advanced cases, an inflammatory reaction composed of macrophages, multinucleated giant cells, and granulation tissue, is observed around the amyloid deposits. Purified amyloid protein is native B2M, and mutations and proteolysis are not believed to be important for its deposition. Plasma levels of B2M are elevated as much as 5-10 times because of the inability of hemodialysis equipment removal of B2M from blood plasma, the duration being very important for B2M amyloid fibrillogenesis. Heparan sulfate proteoglycans, perlecan, is increased at the same sites of amyloid deposits from the early stages. In B2M amyloidosis, an increase of heparan sulfate proteoglycans is observed in the vascular wall and synovium, but in the discs, ligaments and cartilage, there is an increase of chondroitin sulfate proteoglycans predominantly. B2M has an affinity for heparan sulfate proteoglycans, although it is weaker than that for laminin and type IV collagen. This is related to the interactions between negative charges of sulfate groups of proteoglycans and positive charges of basic amino acids in N-terminal side of B2M. Increased cytokines production in the synovium, induced by advanced glycation end products as well as elevated plasma levels, is also linked to inflammatory reactions. Increased expression of matrix metalloproteinases (MMP), especially MMP-1 and -9, is related to the destructive changes of the bone and cartilage. The decrease of plasma levels by high flux membrane and control of inflammatory reactions are very important for prevention of B2M amyloidosis.

Fibroblast growth factors.

Fibroblast growth factors (FGFs) make up a large family of polypeptide growth factors that are found in organisms ranging from nematodes to humans. In vertebrates, the 22 members of the FGF family range in molecular mass from 17 to 34 kDa and share 13-71% amino acid identity. Between vertebrate species, FGFs are highly conserved in both gene structure and amino-acid sequence. FGFs have a high affinity for heparan sulfate proteoglycans and require heparan sulfate to activate one of four cell-surface FGF receptors. During embryonic development, FGFs have diverse roles in regulating cell proliferation, migration and differentiation. In the adult organism, FGFs are homeostatic factors and function in tissue repair and response to injury. When inappropriately expressed, some FGFs can contribute to the pathogenesis of cancer. A subset of the FGF family, expressed in adult tissue, is important for neuronal signal transduction in the central and peripheral nervous systems.

Follistatins neutralize activin bioactivity by inhibition of activin binding to its type II receptors

Follistatin is an activin-binding protein, which inhibits activin bioactivity in several biological systems. In the present study it is demonstrated that preincubation of iodinated activin A with follistatin, purified from porcine follicular fluid, completely abolished the binding of activin to activin type IIA, IIB 2 and IIB 4 receptors, and consequently to activin type IB receptor, transiently transfected in COS cells. Binding of activin A to membrane proteins on the activin-responsive P19 embryonal carcinoma cells was also prevented by this follistatin preparation. The same results were obtained with a carboxy-terminally truncated form of follistatin (FS-288), which is only present in minor amounts in the purified follistatin preparation. Since FS-288 has a high affinity for heparan sulfate proteoglycans on the cell surface, we tested whether membrane-bound FS-288 presents activin A to the different activin receptors, thereby facilitating activin binding. FS-288 did bind to the cell surface of transfected COS cells, but inhibited the binding of activin A to its receptors IIA, IIB 2 and IIB 4. Furthermore, after addition of FS-288 to K562 erythroleukemia cells, the total binding of activin via cell surface-bound FS-288 was increased, whereas the binding of activin A to activin type II and type I receptors present on these cells was inhibited. These findings reveal that different forms of follistatin can neutralize activin bioactivity by interference with binding of activin to all known activin type II receptors, rather than that they inhibit the binding of the type I receptor to the activin/activin type II receptor complex. In addition, our studies indicate that cell surface-associated follistatin cannot present ligand to signalling receptors.

Site-directed mutagenesis of a putative heparin binding domain of avian lipoprotein lipase.

Lipoprotein lipase (LPL) binds to heparin and heparan sulfate proteoglycans. We have employed site-directed mutagenesis to dissect one of the proposed heparin binding domains of avian LPL, which contains the sequence Arg-Lys-Asn-Arg (amino acids 281-284). Various single, double, and triple mutants of chicken LPL were constructed in order to alter the positive charge of this region. The mutant and wild-type cDNAs were subcloned into an expression vector, pRc/CMV, and expressed in Chinese hamster ovary cells. In general, the LPL mutants with a decrease in regional positive charge showed a decrease in affinity for heparin and heparan sulfate proteoglycans. The greatest effect was seen with the triple mutant, LPL 5G, in which all of the positively charged amino acids were altered to neutral residues. On a heparin-Sepharose column, LPL 5G eluted at 0.96 M NaCl compared with 1.35 M for wild-type LPL. This mutant also had the lowest specific activity with 1.5 mu eq fatty acid/micrograms/h for the cell-associated pool and with no detectable activity in the media. Wild-type cells, however, produced a lipase with a specific activity of 12.4 and 13.1 mu eq fatty acid/micrograms/h for cell-associated and media lipase pools, respectively. LPL 5G also showed a decrease in affinity for the heparan sulfate proteoglycans on the cell surface of Chinese hamster ovary cells. In conclusion, the region of avian LPL between Arg281 and Arg284 does appear to be involved in heparin-binding; however, additional regions must be involved since binding was not completely abolished. In addition, specific activity of the cell-associated and secreted LPL is correlated to affinity of the enzyme for heparan sulfate chains.