A large body of evidence supports the involvement of heparan sulfate (HS) proteoglycans in physiological processes such as development and diseases including cancer and neurodegenerative disorders. The role of HS emerges from its ability to interact and regulate the activity of a vast number of extracellular proteins including growth factors and extracellular matrix components. A global view on how protein-HS interactions influence the extracellular proteome and, consequently, cell function is currently lacking. Here, we systematically investigate the functional and structural properties that characterize HS-interacting proteins and the network they form. We collected 435 human proteins interacting with HS or the structurally related heparin by integrating literature-derived and affinity proteomics data. We used this data set to identify the topological features that distinguish the heparin/HS-interacting network from the rest of the extracellular proteome and to analyze the enrichment of gene ontology terms, pathways, and domain families in heparin/HS-binding proteins. Our analysis revealed that heparin/ HS-binding proteins form a highly interconnected network, which is functionally linked to physiological and pathological processes that are characteristic of higher organisms. Therefore, we then investigated the existence of a correlation between the expansion of domain families characteristic of the heparin/HS interactome and the increase in biological complexity in the metazoan lineage. A strong positive correlation between the expansion of the heparin/HS interactome and biosynthetic machinery and organism complexity emerged. The evolutionary role of HS was reinforced by the presence of a rudimentary HS biosynthetic machinery in a unicellular organism at the root of the metazoan lineage.

The cell-extracellular matrix interface is a crowded space whose structure is dependent on macromolecular assemblies that are dynamic in time, molecular composition and location. Signals travel from one cell to another (or to the same cell) by the regulated assembly/disassembly of molecular complexes. These signals can evoke relatively simple biological responses such cell proliferation and migration, but once integrated, they guide cell fate in complex biological phenomena such as embryonic development and organism homeostasis. Heparan sulfate proteoglycans are ubiquitous components of this space and important actors of these processes in all tissue-organized life forms. A key feature of heparan sulfate is its size, 40 nm to 160 nm, which enables it to integrate self-assembling macromolecular structures over substantial length scales. What is the structure of heparan sulfate? Why do we think heparan sulfate is so important? How do we try to explain its activity? What do we know about its interactions? These questions together with a final look to the future are the "menu" of this review.

Heparan sulfate proteoglycans are a ubiquitous component of the extracellular space of complex organisms (1). They are characterized by their size and their plasticity that derives mainly from the complex and dynamically regulated structure of the glycosaminoglycan (GAG) 1 moiety.They participate in the structural organization of the extracellular space (2, 3) and play an active role in molecular networks driving complex biological phenomena such as development (4 -6), inflammation and immune response (7,8), and disease (9). Heparan sulfate proteoglycans exert their functions by interacting with a vast number of protein partners and so regulating their activity (8, 10).In the last decade, a number of techniques have been used to investigate the interaction between proteins and GAGs (for reviews, see . However, despite the identification of more than 200 human heparin-interacting proteins (10, 16), our understanding of the structural features mediating the interaction remains quite poor (10, 13). Some models have been proposed, but they are based on the structural features of only restricted groups or families of heparin-binding proteins (HBPs) (11,17,18). Important limitations for modeling of heparin-binding sites (HBSs) are that many interactions are described solely at a qualitative level and that a three-dimensional structure of the sugar-protein complex is available for less than 10% of the annotated interactions (10). The available data derive mainly from x-ray crystallography (e.g. Refs. 19 and 20), NMR spectroscopy (e.g. Ref. 21), and quantitative biophysics with sitedirected mutagenesis (e.g. Ref. 22) or HBP-derived synthetic peptides (e.g. Ref. 23). Although providing detailed structural and kinetic information about the interaction, all these methods are limited to the study of single protein-sugar interactions, and they cannot be translated into a high throughput format.In 1989 Chang (24) used a lysine-reactive chromophoric reagent to investigate the HBS of antithrombin III (ATIII). ATIII was modified in the presence or absence of heparin, and the colored peptides generated by tryptic digestion were analyzed by RP-HPLC to obtain important insights into the heparin-ATIII interaction. N-Hydroxysuccinimide (NHS) esters have a strong and selective reactivity toward primary amines, in particular -amines on lysines, and their relative stability in neutral/weakly basic aqueous solution makes them compounds of choice for the investigation of HBSs, which are characterized by a high content of basic residues.We developed a rapid and reliable method for the localization of HBSs that can represent an important complement to any structural investigation of heparin-protein interactions because of its low requirement in terms of sample quantity and handling time. The new method, called the "protect and label" strategy, is based on the protection against chemical modification given by heparin/HS to residues located in the HBSs. Thus, an acetyl NHS ester was used to protect residues From the ‡School of Biological Sci...

Cyclic AMP-dependent protein kinase (PKA) enhances regulated exocytosis in neurons and most other secretory cells. To explore the molecular basis of this effect, known exocytotic proteins were screened for PKA substrates. Both cysteine string protein (CSP) and soluble NSF attachment protein-␣ (␣-SNAP) were phosphorylated by PKA in vitro, but immunoprecipitation of cellular ␣-SNAP failed to detect 32 P incorporation. In contrast, endogenous CSP was phosphorylated in synaptosomes, PC12 cells, and chromaffin cells. In-gel kinase assays confirmed PKA to be a cellular CSP kinase, with phosphorylation occurring on Ser 10 . PKA phosphorylation of CSP reduced its binding to syntaxin by 10-fold but had little effect on its interaction with HSC70 or G-protein subunits. Furthermore, an in vivo role for Ser 10 phosphorylation at a late stage of exocytosis is suggested by analysis of chromaffin cells transfected with wild type or non-phosphorylatable mutant CSP. We propose that PKA phosphorylation of CSP could modulate the exocytotic machinery, by selectively altering its availability for protein-protein interactions.Exocytosis is the final stage of the secretory pathway and involves the fusion of secretory vesicles with the plasma membrane in a constitutive or regulated manner (1). In regulated exocytosis, vesicles accumulate in the cytoplasm and only fuse with the plasma membrane upon receipt of an appropriate stimulus (usually, but not always, an increase in intracellular free Ca 2ϩ ). As regulated exocytosis is the basis of chemical transmission in the brain, much research has been devoted to uncovering its molecular mechanism. This has revealed the involvement of a large number of proteins (2, 3), which can be classified into three groups. The first group, proteins involved in vesicle fusion events in all eukaryotes, includes the SNAP 1 receptors, SNAPs, RABs, and the Sec1 family. The second group comprises proteins involved in regulated exocytosis in various cell types and diverse organisms but absent in yeast. This group includes the synaptotagmins and cysteine string proteins (CSP). The third class can be defined as proteins whose role in regulated exocytosis is cell type-specific. An example from this group is the synapsins, which are important modulators of the synaptic vesicle cycle in neurons (4). The complex interactions between the numerous proteins of these classes presumably enables sophisticated fine-tuning of exocytosis to suit the particular physiological needs of each cell type.In addition to the cell type-specific repertoire of exocytotic proteins expressed, further control over the exocytotic mechanism can be exerted post-translationally (5). Indeed, a large number of studies have implicated protein kinases in the modulation of regulated exocytosis from many cell types by using cellpermeable inhibitors or activators, including Ca 2ϩ /calmodulindependent protein kinase II (6, 7), mitogen-activated protein kinase (8), cGMP-dependent protein kinase (9), and tyrosine kinases (8). However, one shortfall of t...

The plasmid pO61 that was isolated from an E. coli genomic DNA library and codes for O6-alkylguanine (O6AG) DNA alkyltransferase (ATase) activity (1) has been further characterised. Subclones of the 9 Kb insert of pO61 showed that the ATase activity was encoded in a 2Kb Pst1 fragment but a partial restriction endonuclease map of this was different to that of the E. coli ada gene that codes for O6-AG and alkylphosphotriester dual ATase protein. Fluorographic analyses confirmed that the molecular weight of the pO61-encoded ATase was 19KDa i.e. similar to that of the O6AG ATase function that is cleaved from the 39KDa ada protein but rabbit polyclonal antibodies to the latter reacted only very weakly with the pO61-encoded protein. A different set of hybridisation signals was produced when E. coli DNA, which had been digested with a variety of restriction endonucleases was probed with 2Kb Pst 1 fragment or the ada gene. These results provided evidence for the existence of a second ATase gene in E. coli. The 2Kb Pst-1 fragment of pO61 was therefore sequenced and an open reading frame (ORF) that would give rise to a 19KDa protein was identified. The derived amino acid sequence of this showed a 93 residue region with 49% homology with the O6AG ATase region of the ada protein and had a pentamer and a heptamer of identical sequence separated by 34 amino acids in both proteins. The pentamer included the alkyl accepting cysteine residue of the ada O6AG ATase. The hydrophobic domains were similarly distributed in both proteins. Shine-Dalgarno, -10 and -35 sequences were identified and the origin of transcription was located by primer extension and S1 nuclease mapping. The amino-terminal amino acid sequence of the protein was as predicted from the ORF.

The interaction between glycosaminoglycans (GAGs) and proteins is important for the regulation of protein transport and activity. Here we present a novel method for the measurement of protein-GAG interactions suitable for high-throughput screening, able to discriminate between the interactions of a protein with GAGs of different structures. Binding of proteins to the GAG heparin, a proxy for sulfated regions of extracellular heparan sulfate, was found to enhance the stability of three test proteins, fibroblast growth factors (FGFs)-1, -2, and -18. Chemically modified heparins and heparin oligosaccharides of different lengths stabilized the three FGFs to different extents, depending on the pattern of sugar binding specificity. The method is based on a differential scanning fluorescence approach. It uses a Sypro Orange dye, which binds to exposed core residues of a denatured protein and results in an increased fluorescence signal. It is convenient, requiring low micromolar amounts of protein and ligand compared to other interaction assays, employing only a real-time polymerase chain reaction (PCR) instrument.

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