<p>Heparan sulfate (HS) is a glycosaminoglycan present in all cell types covalently attached to core proteins forming proteoglycans. HS interacts with different proteins and thereby affects a variety of processes. The biosynthesis of HS takes place in the Golgi network where a complex of the enzymes EXT1 and EXT2 adds N-acetyl glucosamine and glucuronic acid units to the growing chain. The HS chain is <i>N</i>-sulfated by the enzyme <i>N</i>-deacetylase <i>N</i>-sulfotransferase (NDST). <i>N</i>-Sulfation occurs in domains where further modifications (including <i>O</i>-sulfations) take place, giving the chain a complex sulfation pattern.</p><p>In this thesis, new data about the regulation of NDST enzyme activity is presented. By studying NDST1 with active site mutations overexpressed in HEK 293 cells we show that <i>N</i>-deacetylation is the rate-limiting step in HS <i>N</i>-sulfation and that two different NDST molecules can work on the same GlcN unit.</p><p>By analyzing recombinant forms of NDST1 and NDST2 we determined the smallest substrate for <i>N</i>-deacetylation to be an octasaccharide. Importantly, the sulfate donor PAPS was shown to regulate the NDST enzymes to modify the HS chain in domains and that binding of PAPS had a stimulating effect on <i>N</i>-deacetylase activity. </p><p>We could also show that increased levels of NDST1 were obtained when NDST1 was coexpressed with EXT2, while coexpression with EXT1 had the opposite effect. We suggest that EXT2 binds to NDST1, promoting the transport of functional NDST1 to the Golgi network and that EXT1 competes for binding to EXT2. </p><p>Using cell lines overexpressing EXT proteins, it was demonstrated that overexpression of EXT1 increases HS chain length and coexpression of EXT2 results in even longer chains. The enhancing effect of EXT2 was lost when EXT2 was carrying mutations identical to those found in patients with hereditary multiple exostoses, a syndrome characterized by cartilage-capped bony outgrowths at the long bones.</p><p>.</p>

<p>Heparan sulfates (HSs) are linear, negatively charged polysaccharides composed of alternating hexuronic acid (glucuronic acid or iduronic acid) and glucosamine residues that can be substituted to varying degrees with sulfate groups. HS, localized in the extracellular matrix and on the surface of most cells, interacts with a large number of proteins. The actions of HS largely depend on the amount and distribution of its sulfate groups, that provide binding sites for proteins. </p><p>This thesis focuses on the regulation of the structural diversity in HS, in particular the regulation of its 6-<i>O</i>-sulfation patterns that are generated by the combined action of 6-<i>O</i>-sulfotransferases (6OSTs) during biosynthesis, and 6-<i>O</i>-endosulfatases (Sulfs) after completed biosynthesis. In addition, a new model organism is introduced that offers good prospects for investigating the evolutional aspects of HS structural heterogeneity.</p><p>Our studies showed that the three mouse 6OSTs (6OST1-3) exhibit similar substrate specificities <i>in vitro</i>, with minor differences in target preferences. Overexpression of the 6OSTs in cells resulted in increased 6-<i>O</i>-sulfation of both <i>N</i>-sulfated and <i>N</i>-acetylated glucosamine residues. The changes were independent of enzyme isoform but positively correlated to the level of enzyme expressed.</p><p>Quail Sulf1 and Sulf2 enzymes were shown to be cell surface HS 6-<i>O-</i>endosulfatases with preference towards a subset of trisulfated disaccharides within HS chains. The Sulfs contain a “hydrophilic domain” that was shown to be essential for binding of HS, anchorage to the cell surface and endosulfatase activity. QSulf1 was also shown to promote Wnt-Frizzled signaling in cells. </p><p>An HS-like polysaccharide was isolated from the sea anemone <i>Nematostella vectensis</i> and characterized, and all the enzyme families involved in HS biosynthesis and modification in mammalian model systems were also identified. Our results suggest that <i>Nematostella</i> may be a useful tool for understanding the role of evolution in generating HS structural diversity.</p>

Heparan sulfate (HS) is a polysaccharide of glycosaminoglycan type composed of alternating hexuronic acid [either glucuronic acid (GlcA) or iduronic acid (IdoA)] and glucosamine (GlcN) units that can be sulfated in various positions. HS binds to a large number of proteins and these interactions promote many biological processes, including cell adhesion and growth factor signaling. This thesis deals with the structural analysis of short heparan sulfate sequences that mediate binding to fibroblast growth factors FGF1 and FGF2, their receptor FGFR4, and the angiogenesis inhibitor endostatin. Both FGF1 and FGF2 were shown to interact with N-sulfated hexa- and octasaccharide fragments isolated from HS. A pool of HS fragments depleted for FGF1 binding retained the ability to bind FGF2. Changes in 6-O sulfation affected binding to FGF1 but not FGF2, indicating that these proteins bind to distinct HS sequences. All octasaccharides with high affinity for FGF1 contained an internal IdoA2S-GlcNS6S-IdoA2S trisaccharide motif as shown by exoenzyme-based sequence analysis. FGF2 bound to a mono-O-sulfated hexasaccharide with an internal IdoA2S unit, although the affinity was higher for a di-O-sulfated octasaccharide displaying an IdoA2S-GlcNS-IdoA2S trisaccharide motif. FGFR4 was shown to bind the HS analogue heparin with a KD value of 0.3 μM. The interaction between FGFR4 and HS depends on both IdoA2S and GlcNS6S units. Sequence analysis suggested that the number but not the precise location of 6-O-sulfate groups determines affinity. The HS-binding site of endostatin was identified through alanine scanning. Endostatin mutants with reduced affinity for HS were unable to counteract angiogenesis induced by FGF2. The predominant HS motif recognized by endostatin was shown to consist of two N-sulfated domains separated by N-acetylglucosamine units.

Heparan sulfate (HS) is an unbranched and negatively charged polysaccharide of the glycosaminoglycan family, based on the repeated (GlcNAcα1-4GlcAβ1-4) disaccharide structure. The HS backbone is modified by epimerization and sulfation in various positions. HS chains are composed of N-sulfated (NS) domains – predominant locations for further modification steps –, the poorly modified N-acetylated (NA) domains and the alternating NA/NS-domains. HS is present at the cell surface and in the extra-cellular matrix and interacts at these sites with various proteins involved in numerous biological processes, such as angiogenesis. Both pro- and anti-angiogenic proteins can interact with HS and this study was focused on how HS binds to the anti-angiogenic proteins endostatin (ES) and histidine-rich glycoprotein (HRGP) and to pro-angiogenic fibroblast growth factors (FGFs). Here we show that ES recognizes NS-domains in HS spaced by NA-disaccharides, and that binding to ES is abolish through cleavage at these NA-disaccharides. HRGP335, a peptide derived from the His/Pro-rich domain of HRGP is shown to bind to heparin and HS to the same extent as full-size HRGP, in a Zn2+-dependent manner. Moreover, the ability of HRGP to inhibit endothelial cell migration is located to the same region of the protein. We analyzed HS structure in respect to binding to HRGP335 and FGF-2, and show that the ability of HS to bind to those proteins depends on chain length and composition. Finally, the role of HS in FGF–HS–FGF receptor ternary complexes is evaluated using biosynthetic analogs of NS-domains. For stabilization of such complexes the overall sulfation degree of HS seems to play a more pronounced role than the exact distribution of sulfate groups. The results presented in this thesis contribute to a greater understanding of the role of HS in angiogenesis and may provide valuable information for the development of cures against angiogenesis-related disorders.

Heparan sulfate (HS) is a glycosaminoglycan present in all cell types covalently attached to core proteins forming proteoglycans. HS interacts with different proteins and thereby affects a variety of processes. The biosynthesis of HS takes place in the Golgi network where a complex of the enzymes EXT1 and EXT2 adds N-acetyl glucosamine and glucuronic acid units to the growing chain. The HS chain is N-sulfated by the enzyme N-deacetylase N-sulfotransferase (NDST). N-Sulfation occurs in domains where further modifications (including O-sulfations) take place, giving the chain a complex sulfation pattern. In this thesis, new data about the regulation of NDST enzyme activity is presented. By studying NDST1 with active site mutations overexpressed in HEK 293 cells we show that N-deacetylation is the rate-limiting step in HS N-sulfation and that two different NDST molecules can work on the same GlcN unit. By analyzing recombinant forms of NDST1 and NDST2 we determined the smallest substrate for N-deacetylation to be an octasaccharide. Importantly, the sulfate donor PAPS was shown to regulate the NDST enzymes to modify the HS chain in domains and that binding of PAPS had a stimulating effect on N-deacetylase activity. We could also show that increased levels of NDST1 were obtained when NDST1 was coexpressed with EXT2, while coexpression with EXT1 had the opposite effect. We suggest that EXT2 binds to NDST1, promoting the transport of functional NDST1 to the Golgi network and that EXT1 competes for binding to EXT2. Using cell lines overexpressing EXT proteins, it was demonstrated that overexpression of EXT1 increases HS chain length and coexpression of EXT2 results in even longer chains. The enhancing effect of EXT2 was lost when EXT2 was carrying mutations identical to those found in patients with hereditary multiple exostoses, a syndrome characterized by cartilage-capped bony outgrowths at the long bones. .

Heparan sulfates (HSs) are linear, negatively charged polysaccharides composed of alternating hexuronic acid (glucuronic acid or iduronic acid) and glucosamine residues that can be substituted to varying degrees with sulfate groups. HS, localized in the extracellular matrix and on the surface of most cells, interacts with a large number of proteins. The actions of HS largely depend on the amount and distribution of its sulfate groups, that provide binding sites for proteins. This thesis focuses on the regulation of the structural diversity in HS, in particular the regulation of its 6-O-sulfation patterns that are generated by the combined action of 6-O-sulfotransferases (6OSTs) during biosynthesis, and 6-O-endosulfatases (Sulfs) after completed biosynthesis. In addition, a new model organism is introduced that offers good prospects for investigating the evolutional aspects of HS structural heterogeneity. Our studies showed that the three mouse 6OSTs (6OST1-3) exhibit similar substrate specificities in vitro, with minor differences in target preferences. Overexpression of the 6OSTs in cells resulted in increased 6-O-sulfation of both N-sulfated and N-acetylated glucosamine residues. The changes were independent of enzyme isoform but positively correlated to the level of enzyme expressed. Quail Sulf1 and Sulf2 enzymes were shown to be cell surface HS 6-O-endosulfatases with preference towards a subset of trisulfated disaccharides within HS chains. The Sulfs contain a “hydrophilic domain” that was shown to be essential for binding of HS, anchorage to the cell surface and endosulfatase activity. QSulf1 was also shown to promote Wnt-Frizzled signaling in cells. An HS-like polysaccharide was isolated from the sea anemone Nematostella vectensis and characterized, and all the enzyme families involved in HS biosynthesis and modification in mammalian model systems were also identified. Our results suggest that Nematostella may be a useful tool for understanding the role of evolution in generating HS structural diversity.

Heparan sulfate proteoglycans are proteins with long, unbranched heparan sulfate (HS) polysaccharide chains attached to them. They are found on cell surfaces and in basement membranes where they exert their action by interacting with a wide range of enzymes and signaling molecules and are thereby involved in a range of various processes both during embryonic development and in adult physiology. A great part of the biological functionality of proteoglycans can be directly related to the polysaccharide part. HS chains display very variable sulfation patterns where highly sulfated regions are responsible for a large part of the biological activity. The biosynthesis of HS is a complex process in which a number of enzymes are involved. Better comprehension of how this process is regulated could reveal clues to how formation of HS sulfation patterns occurs, and thereby how HS functionality is controlled. This thesis is focusing on regulation of one of the enzymes responsible for HS sulfation, glucosaminyl N-deacetylase/N-sulfotransferase (NDST), in an attempt to understand these mechanisms better. Different aspects of NDST regulation were studied in three projects: I) “Heparin/heparan sulfate biosynthesis: Processive formation of N-sulfated domains”, where the sulfate donor PAPS is shown to influence the manner in which NDST modifies the substrate, affecting the domain structure of the polysaccharide. II) “Heparan sulfate biosynthesis: Characterization of an NDST1 splice variant”, where a splice variant of NDST1 which appears to influence NDST1 protein levels and affect HS structure is described. III) “Heparan sulfate biosynthesis in zebrafish: Five NDST genes with distinct expression patterns during embryonic development”, in which five zebrafish NDSTs were cloned and shown to be expressed in a temporally and spatially regulated manner.

Cervical cancer is the second most common cancer among women worldwide and human papillomavirus (HPV) is a prerequisit for the development of this cancer. HPV belongs to the Papillomaviridae family and infects the basal layer of epithelial cells where it generally progresses into warts or condylomas. HPV can only reproduce in differentiating epithelia and it is therefore difficult to study the natural infection of HPV. More than 100 HPV types exist and they are divided into different genera based on their L1 open reading frame sequence. Most of the HPV types in the alpha-papillomavirus genus infect the mucosal epithelium while HPVs from the beta-papillomavirus genus usually infect cutaneous epithelial cells. Presently, it is not known what decides the anatomical tropism and our aim was to study determinants of this tropism. By using HPV virus like particles (VLP) and pseudovirus we found that VLPs from the two alpha-papillomaviruses HPV-6 and HPV-16 interacted with cell-surface heparan sulfate (HS) for initial attachment. When we labelled HPV VLPs with a fluorescent dye to study internalization HPV-6 was more strongly inhibited than HPV-16. Furthermore, a pseudovirus infection assay demonstrated that the beta-papillomavirus HPV-5 was less dependent on HS for infection than HPV-16. By analyzing the isoelectric point (p1) of the HPV L1 capsid protein we found that alpha HPV types were more positively charged than beta HPV types. Also, HPV-6 had a higher positive charge than HPV-16. Thus, the inhibition of the negatively charged heparin against HPV infection was clearly related to the charge of the HPV L1 capsid. This suggested that the initial interaction could be one of the determinants of tropism although not the sole factor. Lactoferrin is a protein found in milk, saliva, semen, tear fluid and endocervical secretions that has antiviral activities. Both human and bovine lactoferrin inhibited HPV infection but we found no significant differences in inhibition of alpha- and beta-papillomavirus infection. We could however demonstrate that different lactoferricins, small peptide derivates from the N-terminal part of lactoferrin, were able to inhibit HPV infection. This antiviral activity depended on lactoferricin peptide, HPV type and cell origin. The regulation of HPV gene expression in the host cell could also determine HPV tropism. The HPV long control region (LCR) contains cis-responsive elements that regulate HPV transcription and the epithelial tropism of HPV is determined by epithelial specific constitutive enhancers in the LCR. It has been hypothesized that the combination of transcription factors in the host cell determines the cell-type-specific expression. In cells with a skin origin the HPV-5 LCR was twice as efficient in transcriptional activation compared to HPV-16 LCR, while in cervical cells the HPV-16 LCR was almost twice as effective in activating transcription compared to HPV-5 LCR. To conclude, alpha- and beta-papillomaviruses differed regarding their ability to infect cells and regulate viral gene expression. These abilities corresponded with their natural host cells and suggested that HPV anatomical tropism could be determined at several steps in the HPV life cycle.

Heparan sulfate (HS) is a highly sulfated polysaccharide expressed on the cell surface and in the extracellular matrix, interacting with a large number of proteins. HS is implicated in human diseases, including different types of cancer and amyloid diseases such as Alzheimer's disease (AD). The aims of this thesis were to gain deeper insights into AD and cancer progression by elucidating the roles of HS in amyloid-β (Aβ) pathology and hypoxia. The toxic Aβ-peptide is a key molecule in AD due to its ability to aggregate and form amyloid plaques in the brains of diseased patients. It has been reported that HS accumulates with Aβ in these amyloid plaques. We have found that HS is differentially accumulated with Aβ species within the amyloid plaques in the brains of AD patients. We also identified that the HS in the plaques originated from glial cells. Further, we investigated the role of HS in Aβ toxicity using cell models that either lack HS or express abnormal HS. The results show that cell surface HS mediates Aβ internalization and cytotoxicity. Upregulation of heparanase, an endo-glucuronidase that specifically cleaves HS chains, in human cancers increases the potential of tumor cells to metastasize. Spalax, an animal model for hypoxic tolerance, expresses high levels of heparanase. Analysis of HS from different Spalax organs revealed a high sulfation degree and an atypical domain structure, likely modulated by high heparanase expression in the organs. Cells cultured under hypoxic conditions showed a similar HS domain structure and had an increase in heparanase mRNA. We propose that hypoxia-induced heparanase expression is relevant for tumor progression, a process often associated with oxygen deficiency. Altogether, the findings in this thesis are important for future development of therapeutics aiming at interfering with HS functions in AD and cancer.

Angiogenesis is the formation of new blood vessels from pre-existing vasculature and is important during development as well as wound healing and tissue remodeling. Angiogenesis also occurs during pathological conditions such as diabetic retinopathy and cancer. This thesis is centered on the biology of endothelial cells, lining the blood vessels, and myofibroblasts, important for wound healing. We investigated an endothelial cell specific gene, ExoC3l2, and its role in VEGFR2 signaling and migration. EXOC3L2 co-localize with members of the exocyst complex, involved in vesicular transport, as well as VEGFR2. Reducing the level of EXOC3L2 in microvascular endothelial cells results in reduced VEGFR2 signaling and subsequently reduced chemotactic response to VEGF-A. MicroRNA (miRNA) have been shown to be regulators of gene transcription and cell type specific miRNAs have been identified. We investigated two miRNAs, miR-145 and miR-24. miR-145 is expressed in pericytes and fibroblasts but was shown to regulate fli1, an endothelial transcription factor. miR-145 overexpression reduced chemotaxis in both fibroblasts and endothelial cells, as did suppression of the endogenous miR-145 level in fibroblasts. miR-24 in contrast is expressed by endothelial cells and are able to target Ndst1, important for heparan sulfate (HS) sulfation. Sulfation of HS is important for many processes, amongst them growth factor signaling. Overexpression of miR-24 resulted in lower sulfation of HS chains, decreasing the ability of HS to interact with VEGF-A. Overexpressing miR-24 resulted in disturbed chemotaxis, similar to suppressing Ndst1 using siRNA. Myofibroblast recruitment is an important step in wound healing. The myofibroblasts contract the wound, synthesize new extracellular matrix and contribute to revascularization by looping angiogenesis. Maturation from resting fibroblast to myofibroblast is dependent on TGF-β. We found that fibrin fragment E (FnE), a degradation product of fibrin, potentiated the response of fibroblasts to TGF-β thus enhancing TGF-β-induced myofibroblast differentiation. FnE was also found to influence the migration of fibroblasts. These responses are dependent on integrins and toll-like receptors. These findings may serve to further increase the understanding of angiogenesis and wound healing to develop new therapies against pathological conditions.