In vitro biocompatibility and bioactivity of microencapsulated heparan sulfate

The glycosaminoglycan sugar heparan sulfate (HS) is an attractive agent for the repair of bone defects due to its ability to regulate endogenous growth factors. The sustained delivery of HS to the localized wound site over the period of healing which can last for over 1 month may prove advantageous for its therapeutic use. In this study we investigated the encapsulation of HS by the water-in oil-in water (W(1)/O/W(2)) technique in polycaprolactone (PCL) microcapsules as a prolonged delivery device. Encapsulation efficiencies of 70% could be achieved by using a 1:1 mixture of dichloromethane (DCM) and acetone as the solvent in the organic phase, while DCM alone gave poor encapsulation. Although addition of polyvinyl alcohol (PVA) to the drug phase did not affect the size or drug loading of the microcapsules, it did however produce a large change in the morphology and drug distribution, which resulted in different release rates. Release from capsules made with PVA in the drug phase reached 60% after 40 days, while those made with water in the drug phase completed release after 20 days. In vitro biocompatibility studies were performed and detected no increase in cell death in human mesenchymal stem cells (hMSC) or induction of an inflammatory response in macrophages after exposure to release products from HS-loaded microcapsules. The released HS retained its ability to increase the proliferation of hMSC after the encapsulation process. These results indicate that encapsulation of HS by the W(1)/O/W(2) method creates a promising device for the repair of bone tissue.     

The activities of heparan sulfate and its analogue heparin are dictated by biosynthesis, sequence, and conformation

The glycosaminoglycan heparan sulfate (HS), is expressed on the surface of virtually all mammalian cells and is implicated in many crucial biological activities. The activities of HS and its close structural analogue heparin are mediated through interactions with proteins. However, the relationship between structure and activity is not simple, because the structure and conformation of HS and heparin are complex. This review surveys some of the relevant findings in HS/heparin chemistry, biochemistry, and biology.     

Collagen XVIII: a novel heparan sulfate proteoglycan associated with vascular amyloid depositions and senile plaques in Alzheimer's disease brains

Heparan sulfate proteoglycans (HSPGs) may play a role in the formation and persistence of senile plaques and neurofibrillary tangles in Alzheimer's disease brains. Recently, it has been demonstrated that the human extracellular matrix-associated molecule collagen XVIII is the first collagen carrying heparan sulfate side-chains.Two variants of collagen XVIII with both different signal peptides and N-terminal domains have been described and are referred to as the short and long form. To investigate the distribution of these variants we performed an immunohistochemical analysis by using specific well-characterized polyclonal antibodies. Anti-long huXVIII, a polyclonal antibody directed against the long variant of collagen XVIII, weakly stained large cortical and leptomeningeal vessels, whereas small cortical vessels remained unstained. Interestingly, all amyloid-laden vessels and classic senile plaques were strongly stained. Anti-all huXVIII, a polyclonal antibody directed against an epitope common to both collagen XVIII variants, intensely stained all types of cerebral blood vessels, cerebral amyloid angiopathy-affected vessels and classic senile plaques. Collagen XVIII expression was absent in neurofibrillary tangles. We conclude that collagen XVIII is a novel heparan sulfate proteoglycan associated with vascular A beta and classic senile plaques and that at least the long form of collagen XVIII accumulates in amyloid-laden vessels and classic senile plaques.     

Importin beta: a novel autoantigen in human autoimmunity identified by screening random peptide libraries on phage

By screening random peptide libraries (RPLs) with sera of Type 1 diabetes (T1D) patients, we previously identified 5 disease-specific 'mimotopes' displayed on phages (phagotopes). We already characterised 1 phagotope (CH1p), as an epitope of human osteopontin, an autoantigen expressed within the somatostatin cells of human islets. In this paper, we report the characterization of the second phagotope, 195Dyn, by immunohistochemistry, Western Blotting and screening of a human islet cDNA library using rabbit anti-195Dyn antibodies. The 195Dyn mimotope was detected in human islets. The screening of a lambdagt11 cDNA library from human islets has identified a clone, which corresponded to human importin beta. ELISA detected autoantibodies against this protein in sera of around 60% of TD1 patients and in 30% of patients affected by other autoimmune diseases. In summary, RPLs technology proved again successful in identifying another novel autoantigen (importin beta), whose significance in the autoimmune process remains to be fully elucidated.     

Engineering a vascular endothelial growth factor 165-binding heparan sulfate for vascular therapy

The therapeutic use of VEGF₁₆₅ to stimulate blood vessel formation for the treatment of peripheral arterial disease or cardiovascular-related disease has met with limited success. Here we describe an affinity-isolated heparan sulfate glycotherapeutic (HS7^+ve) that binds to, and enhances the bioactivity of, VEGF₁₆₅. Application of HS7^+ve complexed with VEGF₁₆₅ results in enhanced VEGF₁₆₅–VEGFR2 interaction, prolonged downstream pErk1/2 signalling, and increased cell proliferation and tube formation in HUVECs, compared with VEGF₁₆₅ alone. The pro-angiogenic potential of HS7^+ve was further assessed in vivo using the chick embryo chorioallantoic membrane (CAM) assay. Exogenous dosing with HS7^+ve alone significantly enhanced the formation of new blood vessels with potencies comparable to VEGF₁₆₅. These results demonstrate the potential for vascular therapy of glycotherapeutic agents targeted at augmenting the bioactivity of VEGF₁₆₅.     

Production and characterization of a monoclonal antibody against human glomerular heparan sulfate

After immunization with heparan sulfate proteoglycan (HSPG) isolated from human glomeruli, two mouse monoclonal antibodies (mAbs) against heparan sulfate (HS) were obtained. Both mAbs were of the IgM isotype and showed identical specificity. One of these, mAb JM-13 is described in detail. In enzyme-linked immunosorbent assay and Western blotting, reactivity was found with human glomerular basement membrane HSPG and HS. No binding occurred to the core protein of HSPG obtained after removal of HS with trifluoromethanesulfonic acid. In enzyme-linked immunosorbent assay, mAb JM-13 did neither bind to other proteoglycans, nor to other basement membrane components like collagen type IV, laminin, or fibronectin. In indirect immunofluorescence on cryostat sections of human kidneys, a restricted staining of tubular basement membranes was observed along with staining of the vascular basement membranes. In the glomerulus, a weak, fine granular staining was seen along the capillary wall and in the mesangium. MAb JM-13 bound also to the basolateral cell membranes of proximal tubular cells, to the cell membranes of cultured human and rat glomerular visceral epithelial cells, rat mesangial cells, human hepatocytes in culture, and in liver cryostat sections, indicating also a recognition of cell surface-associated HS. Pretreatment of the sections with heparitinase abolished binding of JM-13, whereas treatment with chondroitinase ABC had no effect. Inhibition studies in enzyme-linked immunosorbent assay as well as in indirect immunofluorescence corroborated the HS specificity of mAb JM-13. In conclusion, mAb JM-13 binds to an epitope on the HS chains of glomerular, tubular, and cell surface-associated HSPG.     

Role of heparan sulfate for attachment and entry of tick-borne encephalitis virus

Attachment of the flavivirus tick-borne encephalitis (TBE) virus to different permissive cell lines was investigated by a newly established quantitative assay using fluorescence-labeled virus. Previous work had shown that BHK-21 cell-adapted mutants of TBE virus had acquired potential heparan sulfate (HS) binding sites on the outer surface of protein E. Quantitative analysis of one of these mutants indicated that it attached to HS-expressing cell lines with a 10- to 13-fold higher affinity than wild-type TBE virus strain Neudoerfl. CHO cells deficient in HS synthesis bound less than 5% of the amount of wild-type or mutant virus that could attach to HS-containing CHO cells but were nevertheless found to be highly susceptible to infection with both viruses. Thus, even though HS is a major determinant of TBE virus attachment on HS-expressing cells, our findings suggest the existence of an alternative host cell receptor that is less abundant than HS.     

Nerve injury induces the expression of EXT2, a glycosyltransferase required for heparan sulfate synthesis

Heparan sulfate proteoglycans, which bear long chains of heparan sulfate glycosaminoglycan, play significant roles during embryogenesis, including the formation of the CNS. However, their involvement in nerve regeneration has not yet been clarified. Here, we found that the mRNA expression of EXT2, one of the crucial enzymes for heparan sulfate-glycosaminoglycan synthesis, was markedly up-regulated in injured hypoglossal motor neurons after axotomy. In addition, immunohistochemical staining with an antibody specific for heparan sulfate-glycosaminoglycan chains demonstrated increased expression of heparan sulfate-glycosaminoglycan chains in the injured nucleus. Furthermore, the mRNA expressions of glypican-1 and syndecan-1, which are both well-known heparan sulfate proteoglycans, were prominently up-regulated in injured motor neurons. These results suggest that the biosynthesis of heparan sulfate chains promoted by EXT2 is activated in injured motor neurons, and that glypican-1 and syndecan-1 are potent candidates for heparan sulfate proteoglycans involved in peripheral nerve regeneration.     

Antiviral activity of human lactoferrin: inhibition of alphavirus interaction with heparan sulfate

Human lactoferrin is a component of the non-specific immune system with distinct antiviral properties. We used alphaviruses, adapted to interaction with heparan sulfate (HS), as a tool to investigate the mechanism of lactoferrin's antiviral activity. Lactoferrin inhibited infection of BHK-21 cells by HS-adapted, but not by non-adapted, Sindbis virus (SIN) or Semliki Forest virus (SFV). Lactoferrin also inhibited binding of radiolabeled HS-adapted viruses to BHK-21 cells or liposomes containing lipid-conjugated heparin as a receptor analog. On the other hand, low-pH-induced fusion of the viruses with liposomes, which occurs independently of virus-receptor interaction, was unaffected. Studies involving preincubation of virus or cells with lactoferrin suggested that the protein does not bind to the virus, but rather blocks HS-moieties on the cell surface. Charge-modified human serum albumin, with a net positive charge, had a similar antiviral effect against HS-adapted SIN and SFV, suggesting that the antiviral activity of lactoferrin is related to its positive charge. It is concluded that human lactoferrin inhibits viral infection by interfering with virus-receptor interaction rather than by affecting subsequent steps in the viral cell entry or replication processes.     

Phenylketonuria screening registry as a resource for population genetic studies

Background: Neonatal screening for metabolic diseases, involving samples stored on filter paper (Guthrie spots), provides a potential resource for genetic epidemiological studies. Objective: To develop a method to make these dried blood spots available for large scale genetic epidemiology. Methods: DNA from untraceable Guthrie spots was extracted using a saponin and chelex-100 based method and preamplified by improved primer preamplification. Analyses were done on 38 samples each of fresh, 10, and 25 year old Guthrie spots and the success rate determined for PCR amplification for five amplicon lengths. Results: The method was applicable even on 25 year old samples. The success rate was 100% for 100 bp amplicons and 80% for 396 bp amplicons. Ninety four Guthrie samples were genotyped, including carriers of two different PKU mutations; all carriers were found (six R158Q, four R252W), with no false positives. Finally, 2132 anonymous samples from the Swedish PKU registry were extracted and preamplified and the allele frequencies of APOε4, PPARγ Pro12Ala, and the CCR5 32 bp deletion determined. Local variations in allele frequencies suggested subpopulation structuring. There was a significant difference (p<0.01) in regional allele frequencies for the CCR5 32 bp deletion in the Swedish population. Conclusion: Whole genome amplification makes it feasible to conduct large genetic epidemiological studies using PKU screening registries.     

Improved pattern for genome-based screening identifies novel cell wall-attached proteins in gram-positive bacteria

With a large number of sequenced microbial genomes available, tools for identifying groups or classes of proteins have become increasingly important. Here we present an improved pattern for the identification of cell wall-attached proteins (CWPs), a group of proteins with diverse and important functions in gram-positive bacteria. This tripartite pattern is based on analysis of 65 previously described cell wall-attached proteins and takes into account the three principal requirements for cell wall sorting; a sortase target region (LPXTGX), a membrane-spanning region, and a charged stop-transfer tail. In five different genomes of gram-positive bacteria, the tripartite pattern identified a total of 35 putative CWPs, 19 of which were novel. The specificity and sensitivity of the tripartite pattern are higher than those of the classical pattern, which is based solely on the sortase target region. Several putative CWPs with atypical sortase target regions were identified. In the complete genome of the important human pathogen Streptococcus pyogenes, the tripartite pattern identified 14 putative CWPs. Seven of the putative S. pyogenes proteins were novel, and two of these were a 5' nucleotidase and a pullulanase. This study represents the first whole-genome screening for CWPs, and we conclude that the tripartite pattern is highly suitable for this purpose. Identification of CWPs using this pattern offers important possibilities in the study of the pathogenesis and physiology of gram-positive bacteria.     

Correction: Evaluation of the cost and effectiveness of diverse recruitment methods for a genetic screening study

The original version of this Article contained an error in the undergraduate degree awarded to the author Ian Halim, which was incorrectly given as BS. This has now been corrected to BA in both the PDF.A.nd HTML versions of the Article.     

Glyco-western blotting: biotinylated dermatan sulfate as a probe for the detection of dermatan sulfate binding proteins using western blotting

Dermatan sulfate was biotinylated through the free amino residue of the core protein. The lysates of mouse and human lung culture cells were electrophoresed and blotted to a nitrocellulose membrane. The membrane was blocked successively with bovine serum albumin, avidin and biotin, and then treated with biotinylated dermatan sulfate followed by visualization using alkaline phosphatase conjugated avidin and its substrates. More than 30 bands were observed on the membrane when 1 microgram/ml of biotinylated dermatan sulfate was used. The binding was prevented by an excess of dermatan sulfate but not chondroitin sulfate A or heparan sulfate. Some of the bands resisted washing with high salt concentration buffer. 60 kDa heat shock protein was found to be a dermatan sulfate binding protein upon two dimensional electrophoresis.     

A novel approach for immunization,screening and characterization of selected scFv libraries using membrane fractions of tumor cells

Isolation of cell-surface specific antibodies prerequisites the functional expressing of antigens on intact cells, which are maintained routinely by cell culturing. However, long-term culturing of tumor cells could alter their antigen expression patterns and stable fixation of whole cells is not guaranteed on plastic surfaces during stringent screening procedures. We prepared functional breast cancer cell-membrane fractions that express surface molecules in their native conformation. Specific binding phages were isolated from phage antibody libraries constructed from the spleen messenger RNA of mice immunized with breast cancer cell-membrane fractions. After negative selection on non-mammary carcinoma cells and four rounds of positive selection on breast carcinoma cell lines, phage antibodies were enriched that bound specifically to breast cancer cell lines as confirmed by phage enzyme linked immunosorbent assay (ELISA) using 96-well plates coated with breast cancer cell membranes. The isolated phage antibodies were highly specific for the breast cancer cell line 8701-BC but not on other carcinoma such as the Hodgkin-derived cell line L540Cy as demonstrated by ELISA and flow cytometry. This report describes a rapid and more versatile method for isolating antibody fragments compared to whole cell screening procedures. One single membrane preparation can be stored for at least 15 months at -80 degrees C and used to immunize mice or for screening of antibody libraries. The selection and screening strategy used should be generally applicable to identify novel cell-surface antigens and their corresponding antibodies.     

The use of cationic nanogels to deliver proteins to myeloma cells and primary T lymphocytes that poorly express heparan sulfate

Fusion proteins containing protein transduction domain (PTD) are widely used for intracellular delivery of exogenous proteins. PTD-mediated delivery requires expression of heparan sulfate on the surface of the target cells. However, some of metastatic tumor cells and primary lymphocytes poorly express heparan sulfate. Here we demonstrate that proteins complexed with nanosize hydrogels formed by cationic cholesteryl group-bearing pullulans (cCHP) are efficiently delivered to myeloma cells and primary CD4(+) T lymphocytes probably by induction of macropinocytosis, although these cells are resistant to PTD-mediated protein delivery as a consequence of poor heparan sulfate expression. The anti-apoptotic protein Bcl-xL delivered by cCHP nanogels efficiently blocked apoptosis of these cells, establishing functional regulation of cells by proteins delivered by cCHP nanogels. Thus, cCHP nanogel is a useful tool to deliver proteins for development of new cancer therapy and immune regulation.     

Spatiotemporal distribution of heparan sulfate epitopes during myogenesis and synaptogenesis: a study in developing mouse intercostal muscle.

Formation of a basal lamina (BL) ensheathing developing skeletal muscle cells is one of the earliest events in mammalian skeletal muscle myogenesis. BL-resident heparan sulfate proteoglycans have been implicated in various processes during myogenesis, including synaptic differentiation. However, attention has focused on the proteoglycan protein core, ignoring the glycosaminoglycan moiety mainly because of a lack of appropriate tools. Recently, we selected a panel of anti-heparan sulfate antibodies applied here to study the spatiotemporal distribution of specific heparan sulfate (HS) epitopes during myogenesis. In mouse intercostal muscle at embryonic day (E14), formation of acetylcholine receptor clusters at synaptic sites coincides with HS deposition. Although some HS epitopes show a general appearance throughout the BL, one epitope preferably clusters at synaptic sites but does so only from E16 onward. During elongation and maturation of primary myotubes, a process preceding secondary myotube development, significant changes in the HS epitope constitution of both synaptic and extrasynaptic BL were observed. As a whole, the data presented here strengthen previous observations on developmental regulation by BL components, and add to the putative roles of specific HS epitopes in myogenesis and synaptogenesis.     

The perlecan heparan sulfate proteoglycan mediates cellular uptake of HIV-1 Tat through a pathway responsible for biological activity

Cell surface heparan sulfate proteoglycans (HSPGs) mediate internalization of HIV-1 Tat. Herein, we report that human WiDr cells, which express perlecan but no other HSPGs, can internalize 125I-labeled Tat with minimal lysosomal degradation. Pre-treatment of cells with heparitinase almost completely abolished 125I-Tat surface binding, while the use of an HIV-1 long terminal repeat (LTR) promoter-reporter construct demonstrated that transactivation was potently blocked by pretreatment of cells with heparitinase, indicating an essential role for perlecan in the biologic effects of Tat. We conclude that the perlecan mediates Tat uptake and is required for HIV-1 LTR-directed transactivation in this human cell type.