Proteoglycans (PGs) as the major components of cell surface and extracellular matrix have been actively investigated for well over 40 years. However, there still remains a significant gap in our understanding of the modulation of biosynthesis of a single PG, and regulation of their ex
Proteoglycans are involved in cell signaling, migration and differentiation which make them an important component not only during development but for maintenance throughout adulthood as well. Null mutation of a single proteoglycan could be lethal, as is the loss of aggrecan, a large chondroitin sulfate PG. Aggrecan-null mice (CMD) develop a chondrodysplasia phenotype and die just after birth. Proper modification of glycosaminoglycan (GAG) chains is also crucial for proper biological function. Brachymorphic mice have an undersulfation of GAG chains resulting in dwarfism. The GAG chains associated with the core protein are also vitally important for the functions of PGs. The significance of GAG chains is apparent in heparan sulfate (HS) null mice which result in early embryonic lethality. The role of chondroitin sulfate (CS) GAGs has been highlighted in a recent study involving Colony-stimulation factor-1 (CSF-1) which has three isoforms, one of which is a chondroitin sulfate proteoglycan (CSPG) isoform. CSF-1 null mice have several phenotypes, which are efficiently recovered in transgenic mice expressing the CSPG form of CSF-1. GAGs also play a role in pathology; nude mice injected with Chinese hamster ovary cells with reduced GAG synthesis (15% of total) were found not to be tumorigenic in contrast to wild type cells, implying GAG chains may promote the pathological state.
Proteoglycans and their properly modified GAG chains are involved in many cellular and molecular interactions which are crucial i) during development, ii) necessary for maintenance of a healthy state and/or are iii) subject to modulation under pathological conditions.
The GAG chains of most PGs contain repeating disaccharide units consisting of a hexosamine, either D-glucosamine (GlcN) or D-galactosamine (GalN) and a hexuronic acid, either D-glucuronic acid (GlcA) or L-iduronic acid (IdoA), or in the case of keratan sulfate, the hexuronic acid unit is replaced with a galactose unit. GAG chains are arranged in unbranched, repeating units which can be sulfated in various positions. As indicated earlier, there is extreme diversity among proteoglycans. For example the core protein size can range from 10 kDa to greater than 500 kDa, the number of GAG chains may range from 1 to over 100, and PGs may be hybrid molecules containing GAG chains of more than one type. The most common GAG chains include the galactosaminoglycans, chondroitin sulfate (CS) and dermatan sulfate (DS), and the glucosaminoglycans, heparan sulfate (HS), keratan sulfate (KS) and heparin, as well as hyaluronan (HA) which is a protein-free unsulfated GAG chain.
The sugar and sulfate components of the proteoglycans are transported into the cell through membrane transporters, activated in the cytosol to nucleotide sugars and PAPS, and translocated via transporters into the ER or the Golgi lumen. The energy for the transfer reaction is provided by nucleotide sugars and specificity for donor and acceptor is provided by the enzymes catalyzing the different transferase reactions. GAG biosynthesis is initiated by the transfer of xylose from UDP-xylose to a hydroxyl group of specific serine residues within the core protein, catalyzed by xylosyltransferase. Xylose addition is considered the rate limiting step in GAG biosynthesis, and occurs early in the proteoglycan transport though the secretory pathway, most likely in an ER to golgi intermediate compartment. Xylose is the first sugar in the GAG-protein linkage region (serxylose- galactose-galactose-glucuronic acid), which is common to chondroitin sulfate, dermatan sulfate and heparan sulfate. Following the linkage region, disaccharide units which characterize the GAG chain are added as alternating monosaccharides to the non-reducing end of the elongating chain. Frequently the GAG chains are modified, by phosphorylation of C-2 of xylose, epimerization of glucuronic acid to iduronic acid, and sulfation at various positions catalyzed by specific sulfotransferases.
The importance of heparan sulfate proteoglycans during development has been highlighted in several studies that analyzed the EXT enzymes, which catalyze the initiation and elongation of HS chains. Mice or C. elegans with functionally null homologues of the mammalian EXT1 or EXT2 genes result in embryonic lethality. Drosophila mutants tout-velu (ttv), and sister of tout-velu (sotv), which are the homologues of the mammalian EXT1 and EXT2, respectively, display severe developmental abnormalities as a result of defects in several important signaling pathways which will be discussed later in greater detail. Functional knockout studies have not yet been carried out for the chondroitin sulfate initiating and elongating enzymes. However, studies that have looked at the modification of chondroitin sulfate proteoglycans or the loss of a single CSPG, aggrecan, during development have shown that CSPGs, in addition to HSPGs, are also imperative for proper development. Domowicz et al. found that the loss of aggrecan, a large CSPG, during development in mouse and chick resulted in premature death and severe dwarfism. Cortes et al. found brachymorphic (bm) mice which have undersulfated CSPGs, display chondrodysplasia and shortened limbs as a result of disrupted Ihh signaling. Similarly, a mutation in Drosophila, sulfateless (sfl), in which the mutants have undersulfation of HSPGs results in the disruption of several morphogen signaling pathways. Embryonic development depends on the proper ex