Identification of heparin-binding amino acid residues in antibody HS4C3 with the potential to design antibodies against heparan sulfate domains.

Heparan sulfate (HS) is a linear polysaccharide with high structural and functional diversity. Detection and localization of HS in tissues can be performed using single chain variable fragment (scFv) antibodies. Although several anti-HS antibodies recognizing different sulfation motifs have been identified, little is known about their interaction with HS. In this study the interaction between the scFv antibody HS4C3 and heparin was investigated. Heparin-binding lysine and arginine residues were identified using a protect and label methodology. Site-directed mutagenesis was applied to further identify critical heparin-binding lysine/arginine residues using immunohistochemical and biochemical assays. In addition, computational docking of a heparin tetrasaccharide towards a 3-D homology model of HS4C3 was applied to identify potential heparin-binding sites. Of the 12 lysine and 15 arginine residues within the HS4C3 antibody, 6 and 9, respectively, were identified as heparin-binding. Most of these residues are located within one of the complementarity determining regions (CDR) or in their proximity. All basic amino acid residues in the CDR3 region of the heavy chain were involved in binding. Computational docking showed a heparin tetrasaccharide close to these regions. Mutagenesis of heparin-binding residues reduced or altered reactivity towards HS and heparin. Identification of heparin-binding arginine and lysine residues in HS4C3 allows for better understanding of the interaction with HS and creates a framework to rationally design antibodies targeting specific HS motifs.

Gene expression profiles in mesenchymal stromal cells from bone marrow, adipose tissue and lung tissue of COPD patients and controls.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterized by irreversible lung tissue damage. Novel regenerative strategies are urgently awaited. Cultured mesenchymal stem/stromal cells (MSCs) have shown promising results in experimental models of COPD, but differences between sources may impact on their potential use in therapeutic strategies in patients. AIM: To assess the transcriptome of lung-derived MSCs (LMSCs), bone marrow-derived MSCs (BM-MSC) and adipose-derived MSCs (AD-MSCs) from COPD patients and non-COPD controls. METHODS: We studied differences in gene expression profiles between the MSC-subtypes, as well as between COPD and control using RNA sequencing (RNA-seq). RESULTS: We show that besides heterogeneity between donors, MSCs from different sources have strongly divergent gene signatures. The growth factors FGF10 and HGF were predominantly expressed in LMSCs. MSCs from all sources displayed altered expression profiles in COPD, with most pronounced significantly up- and downregulated genes in MSCs from adipose tissue. Pathway analysis revealed that the most differentially expressed genes in COPD-derived AD-MSCs are involved in extracellular matrix (ECM) binding and expression. In LMSCs, the gene that differed most strongly between COPD and control was CSGALNACT1, an ECM modulating gene. CONCLUSION: Autologous MSCs from COPD patients display abnormalities with respect to their transcriptome, which were surprisingly most profound in MSCs from extrapulmonary sources. LMSCs may be optimally equipped for lung tissue repair because of the expression of specific growth factor genes.

Evaluation and Refinement of Sample Preparation Methods for Extracellular Matrix Proteome Coverage.

The extracellular matrix is a key component of tissues, yet it is underrepresented in proteomic datasets. Identification and evaluation of proteins in the extracellular matrix (ECM) has proved challenging due to the insolubility of many ECM proteins in traditional protein extraction buffers. Here we separate the decellularization and ECM extraction steps of several prominent methods for evaluation under real-world conditions. The results are used to optimize a two-fraction ECM extraction method. Approximately one dozen additional parameters are tested, and recommendations for analysis based on overall ECM coverage or specific ECM classes are given. Compared with a standard in-solution digest, the optimized method yielded a fourfold improvement in unique ECM peptide identifications.

Heparin-resistance in AL amyloidosis: a case report.

BACKGROUND: Non-AT-III mediated heparin-resistance during CPB occurs by complex-forming with heparin-binding proteins. Currently, there are no specific recommendations for non-AT-III mediated heparin-resistance. CASE PRESENTATION: We present a fatal case of a 70-yr-old male-patient undergoing cardiac-surgery in which refractory heparin-resistance was observed. The massive AL amyloidosis found at autopsy is thought to be responsible and illustrates that awareness and knowledge of the etiology and perioperative strategies of non-AT-III mediated heparin-resistance is important. CONCLUSION: For anticoagulation during cardiopulmonary bypass surgery in case of a non-AT-III medicated heparin resistance, we refer to the decision tree added to this manuscript and if necessary to consider direct thrombin inhibitors, such as bivalirudin or argatroban, as it bypasses the complexing pathway.

Oncolytic Virus Therapy with HSV-1 for Hematological Malignancies.

Oncolytic herpes simplex virus type 1 (HSV-1) has been investigated to expand its application to various malignancies. Because hematopoietic cells are resistant to HSV-1, its application to hematological malignancies has been rare. Here, we show that the third generation oncolytic HSV-1, T-01, infected and killed 18 of 26 human cell lines and 8 of 15 primary cells derived from various lineages of hematological malignancies. T-01 replicated at low levels in the cell lines. Viral entry and the oncolytic effect were positively correlated with the expression level of nectin-1 and to a lesser extent 3-O-sulfated heparan sulfate, receptors for glycoprotein D of HSV-1, on tumor cells. Transfection of nectin-1 into nectin-1-negative tumor cells made them susceptible to T-01. The oncolytic effects did not appear to correlate with the expression or phosphorylation of antiviral molecules in the cyclic GMP-AMP (cGAS)-stimulator of interferon genes (STING) and PKR-eIF2α pathways. In an immunocompetent mouse model, intratumoral injection of T-01 into lymphoma induced regression of injected, as well as non-injected, contralateral tumors accompanied by abundant infiltration of antigen-specific CD8+ T cells. These data suggest that intratumoral injection of oncolytic HSV-1 may be applicable to systemic hematological malignancies. Nectin-1 expression may be the most useful biomarker for optimal efficacy.

Regulation of fractone heparan sulfate composition in young and aged subventricular zone neurogenic niches.

Fractones, specialized extracellular matrix structures found in the subventricular zone (SVZ) neurogenic niche, can capture growth factors, such as basic fibroblast growth factor, from the extracellular milieu through a heparin-binding mechanism for neural stem cell (NSC) presentation, which promotes neurogenesis. During aging, a decline in neurogenesis correlates with a change in the composition of heparan sulfate (HS) within fractones. In this study, we used antibodies that recognize specific short oligosaccharides with varying sulfation to evaluate the HS composition in fractones in young and aged brains. To further understand the conditions that regulate 6-O sulfation levels and its impact on neurogenesis, we used endosulfatase Sulf1 and Sulf2 double knockout (DKO) mice. Fractones in the SVZ of Sulf1/2 DKO mice showed immunoreactivity for the HS epitope, suggesting higher 6-O sulfation. While neurogenesis declined in the aged SVZ of both wild-type and Sulf1/2 DKO mice, we observed a larger number of neuroblasts in the young and aged SVZ of Sulf1/2 DKO mice. Together, these results show that the removal of 6-O-sulfation in fractones HS by endosulfatases inhibits neurogenesis in the SVZ. Our findings advance the current understanding regarding the extracellular environment that is best suited for NSCs to thrive, which is critical for the design of future stem cell therapies.

Abnormalities in reparative function of lung-derived mesenchymal stromal cells in emphysema.

Mesenchymal stromal cells (MSCs) may provide crucial support in the regeneration of destructed alveolar tissue (emphysema) in chronic obstructive pulmonary disease (COPD). We hypothesized that lung-derived MSCs (LMSCs) from patients with emphysema are hampered in their repair capacity, either intrinsically or due to their interaction with the damaged microenvironment. LMSCs were isolated from the lung tissue of controls and patients with severe emphysema and characterized at baseline. In addition, LMSCs were seeded onto control and emphysematous decellularized lung tissue scaffolds and assessed for deposition of extracellular matrix (ECM). We observed no differences in surface markers, differentiation/proliferation potential, and expression of ECM genes between control- and COPD-derived LMSCs. Notably, COPD-derived LMSCs displayed lower expression of FGF10 and HGF messenger RNA (mRNA) and hepatocyte growth factor (HGF) and decorin protein. When seeded on control decellularized lung tissue scaffolds, control- and COPD-derived LMSCs showed no differences in engraftment, proliferation, or survival within 2 wk, with similar ability to deposit new matrix on the scaffolds. Moreover, LMSC numbers and the ability to deposit new matrix were not compromised on emphysematous scaffolds. Collectively, our data show that LMSCs from patients with COPD compared with controls show less expression of FGF10 mRNA, HGF mRNA and protein, and decorin protein, whereas other features including the mRNA expression of various ECM molecules are unaffected. Furthermore, COPD-derived LMSCs are capable of engraftment, proliferation, and functioning on native lung tissue scaffolds. The damaged, emphysematous microenvironment as such does not hamper the potential of LMSCs. Thus, specific intrinsic deficiencies in growth factor production by diseased LMSCs may contribute to impaired alveolar repair in emphysema.

A mutant-cell library for systematic analysis of heparan sulfate structure-function relationships.

Heparan sulfate (HS) is a complex linear polysaccharide that modulates a wide range of biological functions. Elucidating the structure-function relationship of HS has been challenging. Here we report the generation of an HS-mutant mouse lung endothelial cell library by systematic deletion of HS genes expressed in the cell. We used this library to (1) determine that the strictly defined fine structure of HS, not its overall degree of sulfation, is more important for FGF2-FGFR1 signaling; (2) define the epitope features of commonly used anti-HS phage display antibodies; and (3) delineate the fine inter-regulation networks by which HS genes modify HS and chain length in mammalian cells at a cell-type-specific level. Our mutant-cell library will allow robust and systematic interrogation of the roles and related structures of HS in a cellular context.

Distinct osmoregulatory responses to sodium loading in patients with altered glycosaminoglycan structure: a randomized cross-over trial.

BACKGROUND: By binding to negatively charged polysaccharides called glycosaminoglycans, sodium can be stored in the body-particularly in the skin-without concurrent water retention. Concordantly, individuals with changed glycosaminoglycan structure (e.g. type 1 diabetes (DM1) and hereditary multiple exostosis (HME) patients) may have altered sodium and water homeostasis. METHODS: We investigated responses to acute (30-min infusion) and chronic (1-week diet) sodium loading in 8 DM1 patients and 7 HME patients in comparison to 12 healthy controls. Blood samples, urine samples, and skin biopsies were taken to investigate glycosaminoglycan sulfation patterns and both systemic and cellular osmoregulatory responses. RESULTS: Hypertonic sodium infusion increased plasma sodium in all groups, but more in DM1 patients than in controls. High sodium diet increased expression of nuclear factor of activated t-cells 5 (NFAT5)-a transcription factor responsive to changes in osmolarity-and moderately sulfated heparan sulfate in skin of healthy controls. In HME patients, skin dermatan sulfate, rather than heparan sulfate, increased in response to high sodium diet, while in DM1 patients, no changes were observed. CONCLUSION: DM1 and HME patients show distinct osmoregulatory responses to sodium loading when comparing to controls with indications for reduced sodium storage capacity in DM1 patients, suggesting that intact glycosaminoglycan biosynthesis is important in sodium and water homeostasis. Trial registration These trials were registered with the Netherlands trial register with registration numbers: NTR4095 ( https://www.trialregister.nl/trial/3933 at 2013-07-29) and NTR4788 ( https://www.trialregister.nl/trial/4645 at 2014-09-12).

Low-GDP, pH-neutral solutions preserve peritoneal endothelial glycocalyx during long-term peritoneal dialysis.

BACKGROUND: During peritoneal dialysis (PD), solute transport and ultrafiltration are mainly achieved by the peritoneal blood vasculature. Glycocalyx lies on the surface of endothelial cells and plays a role in vascular permeability. Low-glucose degradation product (GDP), pH-neutral PD solutions reportedly offer higher biocompatibility and lead to less peritoneal injury. However, the effects on the vasculature have not been clarified. METHODS: Peritoneal tissues from 11 patients treated with conventional acidic solutions (acidic group) and 11 patients treated with low-GDP, pH-neutral solutions (neutral group) were examined. Control tissues were acquired from 5 healthy donors of kidney transplants (control group). CD31 and ratio of luminal diameter to vessel diameter (L/V ratio) were evaluated to identify endothelial cells and vasculopathy, respectively. Immunostaining for heparan sulfate (HS) domains and Ulex europaeus agglutinin-1 (UEA-1) binding was performed to assess sulfated glycosaminoglycans and the fucose-containing sugar chain of glycocalyx. RESULTS: Compared with the acidic group, the neutral group showed higher CD31 positivity. L/V ratio was significantly higher in the neutral group, suggesting less progression of vasculopathy. Both HS expression and UEA-1 binding were higher in the neutral group, whereas HS expression was markedly more preserved than UEA-1 binding in the acidic group. In vessels with low L/V ratio, which were found only in the acidic group, HS expression and UEA-1 binding were diminished, suggesting a loss of glycocalyx. CONCLUSION: Peritoneal endothelial glycocalyx was more preserved in patients treated with low-GDP, pH-neutral solution. The use of low-GDP, pH-neutral solutions could help to protect peritoneal vascular structures and functions.

Growth factor mimetics for skin regeneration: In vitro profiling of primary human fibroblasts and keratinocytes.

Small molecules have gained considerable interest in regenerative medicine, as they can effectively modulate cell fates in a spatiotemporal controllable fashion. A continuous challenge in the field represents genuine mimicry or activation of growth factor signaling with small molecules. Here, we selected and profiled three compounds for their capacity to directly or indirectly activate endogenous FGF-2, VEGF, or SHH signaling events in the context of skin regeneration. Phenotypic and functional analysis of primary skin fibroblasts and keratinocytes revealed unique, cell-specific activity profiles for the FGF-2 mimetic SUN11602 and the putative VEGF mimetic ONO-1301. Whereas SUN11602 exclusively stimulated keratinocyte differentiation, ONO-1301 mainly affected the proliferation and migration behavior of fibroblasts. In each skin cell type, both compounds selectively enhanced the expression of MMP1 and VEGFA. A combined small molecule FGF-2/VEGF mimicry may not only improve angiogenesis-related microcirculation but also reduce early fibrosis while facilitating wound remodeling at later stages. SUN11602 and ONO-1301 represent valuable tools for improving the management of difficult-to-heal wounds, particularly for the design and development of small molecule-functionalized, next-generation, engineered skin substitutes.

A Genome-Wide Haploid Genetic Screen Identifies Heparan Sulfate-Associated Genes and the Macropinocytosis Modulator TMED10 as Factors Supporting Vaccinia Virus Infection.

Vaccinia virus is a promising viral vaccine and gene delivery candidate and has historically been used as a model to study poxvirus-host cell interactions. We employed a genome-wide insertional mutagenesis approach in human haploid cells to identify host factors crucial for vaccinia virus infection. A library of mutagenized HAP1 cells was exposed to modified vaccinia virus Ankara (MVA). Deep-sequencing analysis of virus-resistant cells identified host factors involved in heparan sulfate synthesis, Golgi organization, and vesicular protein trafficking. We validated EXT1, TM9SF2, and TMED10 (TMP21/p23/p24δ) as important host factors for vaccinia virus infection. The critical roles of EXT1 in heparan sulfate synthesis and vaccinia virus infection were confirmed. TM9SF2 was validated as a player mediating heparan sulfate expression, explaining its contribution to vaccinia virus infection. In addition, TMED10 was found to be crucial for virus-induced plasma membrane blebbing and phosphatidylserine-induced macropinocytosis, presumably by regulating the cell surface expression of the TAM receptor Axl.IMPORTANCE Poxviruses are large DNA viruses that can infect a wide range of host species. A number of these viruses are clinically important to humans, including variola virus (smallpox) and vaccinia virus. Since the eradication of smallpox, zoonotic infections with monkeypox virus and cowpox virus are emerging. Additionally, poxviruses can be engineered to specifically target cancer cells and are used as a vaccine vector against tuberculosis, influenza, and coronaviruses. Poxviruses rely on host factors for most stages of their life cycle, including attachment to the cell and entry. These host factors are crucial for virus infectivity and host cell tropism. We used a genome-wide knockout library of host cells to identify host factors necessary for vaccinia virus infection. We confirm a dominant role for heparin sulfate in mediating virus attachment. Additionally, we show that TMED10, previously not implicated in virus infections, facilitates virus uptake by modulating the cellular response to phosphatidylserine.

Construction and evaluation of an antibody phage display library targeting heparan sulfate.

Heparan sulfate (HS) is a linear polysaccharide with high structural diversity. Different HS epitopes have been detected and localized using single chain variable fragment (scFv) antibodies from a 'single pot' phage display library containing a randomized complementarity determining region of the heavy chain (CDR3). In this study, we created a new library containing anti-HS scFvs that all harbor a dp-38 heavy chain segment where the CDR3 region was engineered to contain the XBBXBX heparin binding consensus site (X = any amino acid, B = R, K or H). The library contained ~1.73 × 106 unique antibodies and was biopanned against HS from several sources. The selected antibodies were sequenced and chemically/immunohistologically characterized. A number of 67 anti-HS scFv antibodies were selected, of which 31 contained a XBBXBX CDR3 sequence. There was a clear preference for glycine at the first and proline at the fourth position of the CDR3. The sequence GZZP(R/K)X (Z = R, K or H, but may also contain N, S, or Q) was unusually overrepresented. Selected antibodies reacted with HS/heparin, but not with other glycosaminoglycans. Antibodies reacted differentially with respect to N-, 2-O, or 6-O-desulfated heparin preparations, and showed distinct topologies of HS epitopes in rat kidney sections. The library may be instrumental in the selection of a large pool of HS epitope-specific antibodies, and - since all antibodies differ only in their 6 amino acid CDR region - may be a tool for a rational design of antibodies recognizing specific HS sulfation patterns.

Primary Ovarian Tumors With Lymphogenic and Hematogenic Metastasis Express High MMP-14, Which Colocalizes With Highly Sulfated Chondroitin Sulfate in the Stroma.

Lymphogenic and hematogenic metastases are uncommon in ovarian cancer, especially at presentation. We hypothesized that MMP-14 and MMP-2, CD44, and highly sulfated chondroitin sulfate (CS-E) may be overexpressed in tumors with these metastatic patterns. These molecules are all present in the ovarian tumor microenvironment, wherein they may interact. In an ovarian cancer cohort of 44 patients with metastases in lymph nodes, spleen, and/or liver, the presence of MMP-14, MMP-2, CD44, and CS-E in both the primary tumor and the metastases was determined with immunohistochemistry and related to clinical characteristics. Immunohistochemical expression was found for MMP-14 in all primary tumors as well as in all metastases and for MMP-2 expression in most of the samples. Most primary tumors with synchronous metastases were positive for CS-E, as well as most primary tumors with metachronous lymphogenic metastases. The expression of the MMPs and CS-E in the stroma seemed to colocalize. For CD44 immunohistochemical expression, this relationship was not found. Epithelial MMP-14 on the one hand and stromal CS-E on the other hand seem to be essential players in ovarian cancer with lymphogenic and hematogenic metastases. CD44 expression is not correlated with the other markers. More research on the interaction of these molecules and their role in the process of dissimination of disease is warranted.

A versatile salt-based method to immobilize glycosaminoglycans and create growth factor gradients.

Glycosaminoglycans (GAGs) are known to play pivotal roles in physiological processes and pathological conditions. To study interactions of GAGs with proteins, immobilization of GAGs is often required. Current methodologies for immobilization involve modification of GAGs and/or surfaces, which can be time-consuming and may involve specialized equipment. Here, we use an efficient and low-cost method to immobilize GAGs without any (chemical) modification using highly concentrated salt solutions. A number of salts from the Hofmeister series were probed for their capacity to immobilize heparin and chondroitin-6-sulfate on microtiter plates applying single chain antibodies against GAGs for detection (ELISA). From all salts tested, the cosmotropic salt ammonium sulfate was most efficient, especially at high concentrations (80-100% (v/v) saturation). Immobilized GAGs were bioavailable as judged by their binding of FGF2 and VEGF, and by their susceptibility towards GAG lyases (heparinase I, II and III, chondroitinase ABC). Using 80% (v/v) saturated ammonium sulfate, block and continuous gradients of heparin were established and a gradient of FGF2 was created using a heparin block gradient as a template. In conclusion, high concentrations of ammonium sulfate are effective for immobilization of GAGs and for the establishment of gradients of both GAGs and GAG-binding molecules, which enables the study to the biological roles of GAGs.

CD44 is required for the pathogenesis of experimental crescentic glomerulonephritis and collapsing focal segmental glomerulosclerosis.

A key feature of glomerular diseases such as crescentic glomerulonephritis and focal segmental glomerulosclerosis is the activation, migration and proliferation of parietal epithelial cells. CD44-positive activated parietal epithelial cells have been identified in proliferative cellular lesions in glomerular disease. However, it remains unknown whether CD44-positive parietal epithelial cells contribute to the pathogenesis of scarring glomerular diseases. Here, we evaluated this in experimental crescentic glomerulonephritis and the transgenic anti-Thy1.1 model for collapsing focal segmental glomerulosclerosis in CD44-deficient (cd44-/-) and wild type mice. For both models albuminuria was significantly lower in cd44-/- compared to wild type mice. The number of glomerular Ki67-positive proliferating cells was significantly reduced in cd44-/- compared to wild type mice, which was associated with a reduced number of glomerular lesions in crescentic glomerulonephritis. In collapsing focal segmental glomerulosclerosis, the extracapillary proliferative cellular lesions were smaller in cd44-/- mice, but the number of glomerular lesions was not different compared to wild type mice. For crescentic glomerulonephritis the influx of granulocytes and macrophages into the glomerulus was similar. In vitro, the growth of CD44-deficient murine parietal epithelial cells was reduced compared to wild type parietal epithelial cells, and human parietal epithelial cell migration could be inhibited using antibodies directed against CD44. Thus, CD44-positive proliferating glomerular cells, most likely parietal epithelial cells, are essential in the pathogenesis of scarring glomerular disease.

The reduction of heparan sulphate in the glomerular basement membrane does not augment urinary albumin excretion.

BACKGROUND: Heparan sulphate proteoglycan (HSPG) is present in the glomerular basement membrane (GBM) and is thought to play a major role in the glomerular charge barrier. Reductions and structural alterations of HSPG are observed in different types of kidney diseases accompanied by proteinuria. However, their causal relations remain unknown. METHODS: We generated podocyte-specific exostosin-like 3 gene (Extl3) knockout mice (Extl3KO) using a Cre-loxP recombination approach. A reduction of HSPG was expected in the GBM of these mice, because EXTL3 is involved in its synthesis. Mice were separated into three groups, according to the loads on the glomeruli: a high-protein diet group, a high-protein and high-sodium diet group and a hyperglycaemic group induced by streptozotocin treatment in addition to maintenance on a high-protein and high-sodium diet. The urinary albumin:creatinine ratio was measured at 7, 11, 15 and 19 weeks of age. Renal histology was also investigated. RESULTS: Podocyte-specific expression of Cre recombinase was detected by immunohistochemistry. Moreover, immunofluorescent staining demonstrated a significant reduction of HSPG in the GBM. Electron microscopy showed irregularities in the GBM and effacement of the foot processes in Extl3KO. The values of the urinary albumin:creatinine ratio were within the range of microalbuminuria in all groups and did not significantly differ between the control mice and Extl3KO. CONCLUSIONS: The reduction of HSPG in the GBM did not augment urinary albumin excretion. HSPG's anionic charge appears to contribute little to the glomerular charge barrier.

Heparan Sulfates Support Pyramidal Cell Excitability, Synaptic Plasticity, and Context Discrimination.

Heparan sulfate (HS) proteoglycans represent a major component of the extracellular matrix and are critical for brain development. However, their function in the mature brain remains to be characterized. Here, acute enzymatic digestion of HS side chains was used to uncover how HSs support hippocampal function in vitro and in vivo. We found that long-term potentiation (LTP) of synaptic transmission at CA3-CA1 Schaffer collateral synapses was impaired after removal of highly sulfated HSs with heparinase 1. This reduction was associated with decreased Ca2+ influx during LTP induction, which was the consequence of a reduced excitability of CA1 pyramidal neurons. At the subcellular level, heparinase treatment resulted in reorganization of the distal axon initial segment, as detected by a reduction in ankyrin G expression. In vivo, digestion of HSs impaired context discrimination in a fear conditioning paradigm and oscillatory network activity in the low theta band after fear conditioning. Thus, HSs maintain neuronal excitability and, as a consequence, support synaptic plasticity and learning.

Mutations in Complement Factor H Impair Alternative Pathway Regulation on Mouse Glomerular Endothelial Cells in Vitro.

Complement factor H (FH) inhibits complement activation and interacts with glomerular endothelium via its complement control protein domains 19 and 20, which also recognize heparan sulfate (HS). Abnormalities in FH are associated with the renal diseases atypical hemolytic uremic syndrome and dense deposit disease and the ocular disease age-related macular degeneration. Although FH systemically controls complement activation, clinical phenotypes selectively manifest in kidneys and eyes, suggesting the presence of tissue-specific determinants of disease development. Recent results imply the importance of tissue-specifically expressed, sulfated glycosaminoglycans (GAGs), like HS, in determining FH binding to and activity on host tissues. Therefore, we investigated which GAGs mediate human FH and recombinant human FH complement control proteins domains 19 and 20 (FH19-20) binding to mouse glomerular endothelial cells (mGEnCs) in ELISA. Furthermore, we evaluated the functional defects of FH19-20 mutants during complement activation by measuring C3b deposition on mGEnCs using flow cytometry. FH and FH19-20 bound dose-dependently to mGEnCs and TNF-α treatment increased binding of both proteins, whereas heparinase digestion and competition with heparin/HS inhibited binding. Furthermore, 2-O-, and 6-O-, but not N-desulfation of heparin, significantly increased the inhibitory effect on FH19-20 binding to mGEnCs. Compared with wild type FH19-20, atypical hemolytic uremic syndrome-associated mutants were less able to compete with FH in normal human serum during complement activation on mGEnCs, confirming their potential glomerular pathogenicity. In conclusion, our study shows that FH and FH19-20 binding to glomerular endothelial cells is differentially mediated by HS but not other GAGs. Furthermore, we describe a novel, patient serum-independent competition assay for pathogenicity screening of FH19-20 mutants.

A potential role for chondroitin sulfate/dermatan sulfate in arm regeneration in Amphiura filiformis.

Glycosaminoglycans (GAGs), such as chondroitin sulfate (CS) and dermatan sulfate (DS) from various vertebrate and invertebrate sources are known to be involved in diverse cellular mechanisms during repair and regenerative processes. Recently, we have identified CS/DS as the major GAG in the brittlestar Amphiura filiformis, with high proportions of di- and tri-O-sulfated disaccharide units. As this echinoderm is known for its exceptional regeneration capacity, we aimed to explore the role of these GAG chains during A. filiformis arm regeneration. Analysis of CS/DS chains during the regeneration process revealed an increase in the proportion of the tri-O-sulfated disaccharides. Conversely, treatment of A. filiformis with sodium chlorate, a potent inhibitor of sulfation reactions in GAG biosynthesis, resulted in a significant reduction in arm growth rates with total inhibition at concentrations higher than 5 mM. Differentiation was less impacted by sodium chlorate exposure or even slightly increased at 1-2 mM. Based on the structural changes observed during arm regeneration we identified chondroitin synthase, chondroitin-4-O-sulfotransferase 2 and dermatan-4-O-sulfotransferase as candidate genes and sought to correlate their expression with the expression of the A. filiformis orthologue of bone morphogenetic factors, AfBMP2/4. Quantitative amplification by real-time PCR indicated increased expression of chondroitin synthase and chondroitin-4-O-sulfotransferase 2, with a corresponding increase in AfBMP2/4 during regeneration relative to nonregenerating controls. Our findings suggest that proper sulfation of GAGs is important for A. filiformis arm regeneration and that these molecules may participate in mechanisms controlling cell proliferation.