Sulfatase activities towards the regulation of cell metabolism and signaling in mammals

In higher vertebrates, sulfatases belong to a conserved family of enzymes that are involved in the regulation of cell metabolism and in developmental cell signaling. They cleave the sulfate from sulfate esters contained in hormones, proteins, and complex macromolecules. A highly conserved cysteine in their active site is post-translationally converted into formylglycine by the formylglycine-generating enzyme encoded by SUMF1 (sulfatase modifying factor 1). This post-translational modification activates all sulfatases. Sulfatases are extensively glycosylated proteins and some of them follow trafficking pathways through cells, being secreted and taken up by distant cells. Many proteoglycans, glycoproteins, and glycolipids contain sulfated carbohydrates, which are sulfatase substrates. Indeed, sulfatases operate as decoding factors for a large amount of biological information contained in the structures of the sulfated sugar chains that are covalently linked to proteins and lipids. Modifications to these sulfate groups have pivotal roles in modulating specific signaling pathways and cell metabolism in mammals.     

Lecticans: organizers of the brain extracellular matrix

Lecticans are a family of chondroitin sulfate proteoglycans, encompassing aggrecan, versican, neurocan and brevican. These proteoglycans are characterized by the presence of ahyaluronan-binding domain and a C-type lectin domain in their core proteins. Through these domains, lecticans interact with carbohydrate and protein ligands in the extracellular matrix and act as linkers of these extracellular matrix molecules. In adult brain, lecticans are thought to interact with hyaluronan and tenascin-R to form a ternary complex. We propose that the hyaluronan-lectican-tenascin-R complex constitutes the core assembly of the adult brain extracellular matrix, which is found mainly in pericellular spaces of neurons as ‘perineuronal nets’. Received 27 September 1999; accepted 26 October 1999     

Proteoglycans of basement membranes

Proteoglycans carrying either heparan sulfate and/or chondroitin sulfate side chains are typical constituents of basement membranes. The most prominent proteoglycan (perlecan) consists of a 400–500 kDa core protein and three heparan sulfate chains. Electron microscopy and cDNA sequencing show a complex and elongated domain structure for the core protein which in part is homologous to that of the laminin A chain. This structure may be varied by alternative splicing and proteolysis. Integration into basement membranes probably occurs by heparan sulfate binding to laminin and collagen IV, core protein binding to nidogen and by limited self assembly. The proteoglycan is in addition a cell-adhesive protein which is recognized by 1 integrins. Several more proteoglycans with smaller core proteins (10–160 kDa) apparently exist in basement membranes but are less well characterized. Biological functions include control of filtration through basement membranes and binding of growth factors and protease inhibitors.     

Circular proteoglycans from sponges: first members of the spongican family

Species-specific cell adhesion in marine sponges is mediated by a new family of modular proteoglycans whose general supramolecular structure resembles that of hyalectans. However, neither their protein nor their glycan moieties have significant sequence homology to other proteoglycans, despite having protein subunits equivalent to link proteins and to proteoglycan monomer core proteins, and glycan subunits equivalent to hyaluronan and to the glycosaminoglycans of hyalectans. In some species, these molecular components are assembled into a structure with a circular core formed by the link protein- and hyaluronan-like subunits. Besides their involvement in cell adhesion, these sponge proteoglycans, for which we propose the term spongicans, participate in signal transduction processes and are suspected to play a role in sponge self-nonself recognition. Their in vivo roles and the mild methods used to purify large amounts of functionally active spongicans make them ideal models to study the functions and possible new applications of proteoglycans in biomedical research. Received 21 May 2002; received after revision 5 July 2002; accepted 10 July 2002 RID="*" ID="*"Corresponding author.     

Cell surface heparan sulfate proteoglycans and lipoprotein metabolism

Cell surface heparan sulfate proteoglycans are involved in several aspects of the lipoprotein metabolism. Most of the biological activities of these proteoglycans are mediated via interactions of their heparan sulfate moieties with various protein ligands, including lipoproteins and lipases. The binding of lipoproteins to heparan sulfate is largely determined by their apoprotein composition, and apoproteins B and E display the highest affinity for heparan sulfate. Interactions of lipoproteins with heparan sulfate are important for the cellular uptake and turnover of lipoproteins, in part by enhancing the accessibility of lipoproteins to lipoprotein receptors and lipases. Apoprotein B may interact with receptors without involving heparan sulfate. Heparan sulfate has been further implicated in presentation and stabilization of lipoprotein lipase and hepatic lipase on cell surfaces and in the transport of lipoprotein lipase from extravascular cells to the luminal surface of the endothelia. In atherosclerosis, heparan sulfate is intimately involved in several events important to the pathophysiology of the disease. Heparan sulfate thus binds and regulates the activity of growth factors, cytokines, superoxide dismutase and antithrombin, which contribute to aberrant cell proliferation, migration and matrix production, scavenging of reactive oxygen radicals and thrombosis. In this review we discuss the various roles of heparan sulfate proteoglycans in vascular biology, with emphasis on interactions of heparan sulfate with lipoproteins and lipases and the molecular basis of such interactions.     

Amyloid precursor protein and its homologues: a family of proteolysis-dependent receptors

The Alzheimer’s amyloid precursor protein (APP) belongs to a conserved gene family that also includes the mammalian APLP1 and APLP2, the Drosophila APPL, and the C. elegans APL-1. The biological function of APP is still not fully clear. However, it is known that the APP family proteins have redundant and partly overlapping functions, which demonstrates the importance of studying all APP family members to gain a more complete picture. When APP was first cloned, it was speculated that it could function as a receptor. This theory has been further substantiated by studies showing that APP and its homologues bind both extracellular ligands and intracellular adaptor proteins. The APP family proteins undergo regulated intramembrane proteolysis (RIP), generating secreted and cytoplasmic fragments that have been ascribed different functions. In this review, we will discuss the APP family with focus on biological functions, binding partners, and regulated processing.     

Distribution of tightened end fragments of globular proteins statistically matches that of topohydrophobic positions: towards an efficient punctuation of protein folding?

Using a set of 372 proteins representative of a variety of 56 distinct globular folds, a statistical correlation was observed between two recently revealed features of protein structures: tightened end fragments or 'closed loops', i. e. sequence fragments that are able in three-dimensional (3D) space to nearly close their ends (a current parameter of polymer physics), and 'topohydrophobic positions', i. e. positions always occupied in 3D space by strong hydrophobic amino acids for all members of a fold family. Indeed, in sequence space, the distribution of preferred lengths for tightened end fragments and that for topohydrophobic separation match. In addition to this statistically significant similarity, the extremities of these 'closed loops' may be preferentially occupied by topohydrophobic positions, as observed on a random sample of various folds. This observation may be of special interest for sequence comparison of distantly related proteins. It is also important for the ab initio prediction of protein folds, considering the remarkable topological properties of topohydrophobic positions and their paramount importance within folding nuclei. Consequently, topohydrophobic positions locking the 'closed loops' belong to the deep cores of protein domains and might have a key role in the folding process. Received 1 February 2001; accepted 7 February 2001     

Small proteoglycans

In this review the structure and functions of two non-related proteoglycan families are discussed. One family represents a group of extracellular matrix macromolecules characterized by core proteins with leucine-rich repeat motifs. Within this family special attention is given to those members which carry chondroitin or dermatan sulfate glycosaminoglycan chains. The second family is characterized by repeat sequences of serine and glycine. Their members are products of a single core protein gene and are characteristic constituents of secretory vesicles in cells of the haematopoietic lineage.     

Perforin and its role in T lymphocyte-mediated cytolysis

The killing mediated by cytotoxic T lymphocytes (CTL) represents an important mechanism in the immune defence against tumors and virus infections. The lytic mechanism has been proposed to consist of a polarized secretion of granule-stored molecules, occurring on effector-target cell contact. By electron microscopy, membrane deposited, pore-like lesions are detected on the target cell membrane during cytolysis by CTL. These structures resembled strikingly pores formed during complement attack.Granules of CTL isolated by nitrogen cavitation and Percoll gradient centrifugation were shown to retain cytotoxic activity. Further purification of proteins stored in these granules led to the discovery of a membranolytic protein named perforin which was capable of polymerizing into pore-like structures. In addition to this cytolytic protein, a set of serine esterases was found as well as lysosomal enzymes and proteoglycans, whose function are not yet clearly defined. The role of perforin in the cytotoxic process is currently being explored by ablating the active gene in mice.     

Perforin and its role in T lymphocyte-mediated cytolysis.

The killing mediated by cytotoxic T lymphocytes (CTL) represents an important mechanism in the immune defence against tumors and virus infections. The lytic mechanism has been proposed to consist of a polarized secretion of granule-stored molecules, occurring on effector-target cell contact. By electron microscopy, membrane deposited, pore-like lesions are detected on the target cell membrane during cytolysis by CTL. These structures resembled strikingly pores formed during complement attack. Granules of CTL isolated by nitrogen cavitation and Percoll gradient centrifugation were shown to retain cytotoxic activity. Further purification of proteins stored in these granules led to the discovery of a membranolytic protein named perforin which was capable of polymerizing into pore-like structures. In addition to this cytolytic protein, a set of serine esterases was found as well as lysosomal enzymes and proteoglycans, whose function are not yet clearly defined. The role of perforin in the cytotoxic process is currently being explored by ablating the active gene in mice.     

Extracellular matrix components associated with remodeling processes in brain

In the central nervous system, various extracellular matrix components have been identified which are strongly expressed during development and in most areas of the brain down-regulated during maturation. Examples are tenascin-C, neurocan and hyaluronan. While tenascin-C is well known to be associated with morphogenic events and the active contribution of hyaluronan to various physiological processes is increasingly acknowledged, neurocan belongs to a class of molecules thought to be generally more associated with barrier functions: chondroitin sulfate proteoglycans. Consideration of these and related molecules and their processing in the context of the general organization of the brain extracellular matrix, their changes during brain maturation and their implication in different types of remodeling processes in adult brain, like normal and pathological synaptic plasticity, inflammatory and dementia-associated diseases and gliomas, may indicate that components of the extracellular matrix could provide valuable early information about the pathological state of the brain.Received 29 January 2004; received after revision 25 March 2004; accepted 2 April 2004     

Effect of olfactory ensheathing cells on reactive astrocytes <Emphasis Type="Italic">in vitro</Emphasis>

Olfactory ensheathing cells have been used in several studies to promote repair in the injured spinal cord. However, cellular interaction between olfactory ensheathing cells and glial cells induced to be reactive in the aftermath of injury site has not been investigated. Using an in vitro model of astrogliosis, we show that reactive astrocytes expressed significantly less glial fibrillary acidic protein (GFAP) when cultured both in direct contact with olfactory ensheathing cells and when the two cell types were separated by a porous membrane. Immunofluorescence staining also suggested that reactive astrocytes showed decreased chondroitin sulfate proteoglycans in the presence of olfactory ensheathing cells, although the reduction was not statistically significant. No down-regulation of GFAP was observed when reactive astrocytes were similarly cultured with Schwann cells. Cell viability assay and bromodeoxyuridine uptake showed that proliferation of reactive astrocytes was significantly increased in the presence of olfactory ensheathing cells and Schwann cells. Received 27 February 2007; received after revision 30 March 2007; accepted 3 April 2007     

A dynamic view of peptides and proteins in membranes

Biological membranes are highly dynamic supramolecular arrangements of lipids and proteins, which fulfill key cellular functions. Relatively few high-resolution membrane protein structures are known to date, although during recent years the structural databases have expanded at an accelerated pace. In some instances the structures of reaction intermediates provide a stroboscopic view on the conformational changes involved in protein function. Other biophysical approaches add dynamic aspects and allow one to investigate the interactions with the lipid bilayers. Membrane-active peptides fulfill many important functions in nature as they act as antimicrobials, channels, transporters or hormones, and their studies have much increased our understanding of polypeptide-membrane interactions. Interestingly several proteins have been identified that interact with the membrane as loose arrays of domains. Such conformations easily escape classical high-resolution structural analysis and the lessons learned from peptides may therefore be instructive for our understanding of the functioning of such membrane proteins. Received 11 March 2008; received after revision 2 May 2008; accepted 5 May 2008     

Immunoglobulin light chains, glycosaminoglycans, and amyloid

Immunoglobulin light chains are the precursor proteins for fibrils that are formed during primary amyloidosis and in amyloidosis associated with multiple myeloma. As found for the approximately 20 currently described forms of focal, localized, or systemic amyloidoses, light chain-related fibrils extracted from physiological deposits are invariably associated with glycosaminoglycans, predominantly heparan sulfate. Other amyloid-related proteins are either structurally normal, such as beta2-microglobulin and islet amyloid polypeptide, fragments of normal proteins such as serum amyloid A protein or the precursor protein of the beta peptide involved in Alzheimer's disease, or are inherited forms of single amino acid variants of a normal protein such as found in the familial forms of amyloid associated with transthyretin. In contrast, the primary structures of light chains involved in fibril formation exhibit extensive mutational diversity rendering some proteins highly amyloidogenic and others non-pathological. The interactions between light chains and glycosaminoglycans are also affected by amino acid variation and may influence the clinical course of disease by enhancing fibril stability and contributing to resistance to protease degradation. Relatively little is currently known about the mechanisms by which glycosaminoglycans interact with light chains and light-chain fibrils. It is probable that future studies of this uniquely diverse family of proteins will continue to shed light on the processes of amyloidosis, and contribute as well to a greater understanding of the normal physiological roles of glycosaminoglycans.     

CCN1/CYR61: the very model of a modern matricellular protein

CCN1 (CYR61) is a dynamically expressed, multifunctional matricellular protein that plays essential roles in cardiovascular development during embryogenesis, and regulates inflammation, wound healing and fibrogenesis in the adult. Aberrant CCN1 expression is associated with myriad pathologies, including various cancers and diseases associated with chronic inflammation. CCN1 promotes diverse and sometimes opposing cellular responses, which can be ascribed, as least in part, to disparate activities mediated through its direct binding to distinct integrins in different cell types and contexts. Accordingly, CCN1 promotes cell proliferation, survival and angiogenesis by binding to integrin α_vβ₃, and induces apoptosis and senescence through integrin α₆β₁ and heparan sulfate proteoglycans. The ability of CCN1 to trigger the accumulation of a robust and sustained level of reactive oxygen species underlies some of its unique activities as a matrix cell-adhesion molecule. Emerging studies suggest that CCN1 might be useful as a biomarker or therapeutic target in certain diseases.     

<Emphasis Type="Italic">Dictyostelium</Emphasis> dynamin B modulates cytoskeletal structures and membranous organelles

Dictyostelium discoideum cells produce five dynamin family proteins. Here, we show that dynamin B is the only member of this group of proteins that is initially produced as a preprotein and requires processing by mitochondrial proteases for formation of the mature protein. Our results show that dynamin B-depletion affects many aspects of cell motility, cell-cell and cell-surface adhesion, resistance to osmotic shock, and fatty acid metabolism. The mature form of dynamin B mediates a wide range and unique combination of functions. Dynamin B affects events at the plasma membrane, peroxisomes, the contractile vacuole system, components of the actin-based cytoskeleton, and cell adhesion sites. The modulating effect of dynamin B on the activity of the contractile vacuole system is unique for the Dictyostelium system. Other functions displayed by dynamin B are commonly associated with either classical dynamins or dynamin-related proteins.     

Heparan sulfate-protein interactions: therapeutic potential through structure-function insights

Heparin and the related glycosaminoglycan, heparan sulfate, bind a myriad of proteins. The structural diversity of heparin and heparan sulfates is enormous, but differences in the conformational flexibility of the monosaccharide constituents add extra complexity and may influence protein binding. Silencing genes for heparin/ heparan sulfate biosynthetic enzymes profoundly affects mammalian development. Thus, altering the structure of heparan sulfate chains can alter protein binding and embryo development. Different heparan sulfate structures are located in particular tissue sites, and these structures are recognised by different sets of proteins. Regulation of certain heparan sulfate-protein interactions by pH or cations is described. Heparin/heparan sulfate structures are viewed as potential therapeutics for a variety of diseases. An understanding at the molecular and functional levels of the specificity and affinity of heparan sulfate-protein interactions is crucial for designing heparin-inspired drugs. How the development of synthesis techniques is facilitating structure-function analyses and drug development is discussed.Received 6 July 2004; received after revision 16 September 2004; accepted 28 September 2004     

Emerging roles of the oxysterol-binding protein family in metabolism, transport, and signaling

OSBP (oxysterol-binding protein) and ORPs (OSBP-related proteins) constitute an enigmatic eukaryotic protein family that is united by a signature domain that binds oxysterols, sterols, and possibly other hydrophobic ligands. The human genome contains 12 OSBP/ORP family members genes, while that of the budding yeast Saccharomyces cerevisiae encodes seven OSBP homologues (Osh). Of these, Osh4 (also referred to as Kes1) has been the most widely studied to date. Recently, three-dimensional crystal structures of Osh4 with and without sterols bound within the core of the protein were determined. The core consists of 19 anti-parallel β-sheets that form a near-complete β-barrel. Recent work has suggested that Osh proteins facilitate the non-vesicular transport of sterols in vivo and in vitro, while other evidence supports a role for Osh proteins in the regulation of vesicular transport and lipid metabolism.This article will review recent advances in the study of ORP/Osh proteins and will discuss future research issues regarding the ORP/Osh family. Received 17 July 2007; received after revision 14 August 2007; accepted 12 September 2007     

Isolation and purification of proteoglycans

Purification of a protein typically involves development of a quantitative assay to track protein integrity (e.g. enzyme activity) during subsequent isolation steps. The generalized procedure involves choosing the source of the protein, defining extraction conditions, developing bulk purification methods followed by refined, more selective methods. The purification of proteoglycans is often complicated by a) limited source quantities, b) necessity of chaotropic solvents for efficient extraction, c) their large molecular size and d) lack of defined functions to enable purity (i.e. activity, conformation) to be assessed. Because the usual goal of proteoglycan purification is physical characterization (intact molecular weight, core protein and glycosaminoglycan class and size), the problems of a suitable assay and/or native conformation are avoided. The assay for tracking proteoglycan isolation typically utilizes uronic acid content or radiolabel incorporation as a marker. Once extracted from their cellular/extracellular environment, proteoglycans can be isolated by density gradient centrifugation and/or column chromatography techniques. Recent advances in the composition of chromatographic supports have enabled the application of ion-exchange, gel permeation, hydrophobic interaction and affinity chromatography resins using efficient high-pressure liquid chromatography to proteoglycan purification.