Biosynthesis and function of chondroitin sulfate

Background

Chondroitin sulfate proteoglycans (CSPGs) are principal pericellular and extracellular components that form regulatory milieu involving numerous biological and pathophysiological phenomena. Diverse functions of CSPGs can be mainly attributed to structural variability of their polysaccharide moieties, chondroitin sulfate glycosaminoglycans (CS-GAG). Comprehensive understanding of the regulatory mechanisms for CS biosynthesis and its catabolic processes is required in order to understand those functions.

Scope of review

Here, we focus on recent advances in the study of enzymatic regulatory pathways for CS biosynthesis including successive modification/degradation, distinct CS functions, and disease phenotypes that have been revealed by perturbation of the respective enzymes in vitro and in vivo.

Major conclusions

Fine-tuned machineries for CS production/degradation are crucial for the functional expression of CS chains in developmental and pathophysiological processes.

General significance

Control of enzymes responsible for CS biosynthesis/catabolism is a potential target for therapeutic intervention for the CS-associated disorders.     

Reduced Expression of EXTL2, a Member of the Exostosin (EXT) Family of Glycosyltransferases,in Human Embryonic Kidney 293 Cells Results in Longer Heparan Sulfate Chains

Heparan sulfate proteoglycans are ubiquitously located on cell surfaces and in the extracellular matrices. The negatively charged heparan sulfate chains interact with a multitude of different proteins, thereby influencing a variety of cellular and developmental processes, for example cell adhesion, migration, tissue morphogenesis, and differentiation. The human exostosin (EXT) family of genes contains five members: the heparan sulfate polymerizing enzymes, EXT1 and EXT2, and three EXT-like genes, EXTL1, EXTL2, and EXTL3. EXTL2 has been ascribed activities related to the initiation and termination of heparan sulfate chains. Here we further investigated the role of EXTL2 in heparan sulfate chain elongation by gene silencing and overexpression strategies. We found that siRNA-mediated knockdown of EXTL2 in human embryonic kidney 293 cells resulted in increased chain length, whereas overexpression of EXTL2 in the same cell line had little or no effect on heparan sulfate chain length. To study in more detail the role of EXTL2 in heparan sulfate chain elongation, we tested the ability of the overexpressed protein to catalyze the in vitro incorporation of N-acetylglucosamine and N-acetylgalactosamine to oligosaccharide acceptors resembling unmodified heparan sulfate and chondroitin sulfate precursor molecules. Analysis of the generated products revealed that recombinant EXTL2 showed weak ability to transfer N-acetylgalactosamine to heparan sulfate precursor molecules but also, that EXTL2 exhibited much stronger in vitro N-acetylglucosamine-transferase activity related to elongation of heparan sulfate chains.     

Exostosin-like 2 regulates FGF2 signaling by controlling the endocytosis of FGF2

Background

Heparan sulfate proteoglycans are ubiquitously expressed on cell surfaces and in extracellular matrices, and are engaged in heparin-binding growth factor-related signal transduction. Thus, changes in the amounts, structures, and chain lengths of heparan sulfate have profound effects on aspects of cell growth controlled by heparin-binding growth factors such as FGF2. Exostosin glycosyltransferases (EXT1, EXT2, EXTL1, EXTL2, and EXTL3) control heparan sulfate biosynthesis, and the expression levels of their genes regulate the amounts, chain lengths, and sulfation patterns of heparan sulfate. Unlike EXT1, EXT2, and EXTL3, EXTL2 functions chain termination of heparan sulfate. Here, we examined the importance of EXTL2 in FGF2-dependent signaling.

Analysis of novel alleles of brother of tout‐velu,the drosophila ortholog of human EXTL3 using a newly developed FRT42D ovoD chromosome

The FLP/FRT system permits rapid phenotypic screening of homozygous lethal mutations in the context of a viable mosaic fly. Combining this system with ovo^D dominant female‐sterile transgenes enables efficient production of embryos derived from mutant germline clones lacking maternal contribution from a gene of interest. Two distinct sets of FRT chromosomes, carrying either the mini‐white (w ^{+ mW.hs}), or rosy (ry⁺) and neomycin (neo^R) transgenes are in common use. Parallel ovo^D lines were developed using w ^{+ mW.hs} FRT insertions on the X and chromosomes 2R and 3L, as well as ry⁺, neo^R FRT insertions on 2L and 3R. Consequently, mutations isolated on the X, 2R and 3L chromosomes in a ry⁺, neo^R FRT background, are not amenable to germline clonal analysis without labor‐intensive recombination onto chromosome arms containing a w ^{+ mW.hs} FRT. Here we report the creation of a new ovo^D line for the ry⁺, neo^R FRT insertion at position FRT42D on chromosome 2R, through induced recombination in males. To establish the developmental relevance of this reagent we characterized the maternal‐effect phenotypes of novel brother of tout‐velu alleles generated on an FRT42D chromosome in a somatic mosaic screen. We find that an apparent null mutation that causes severe defects in somatic tissues has a much milder effect on embryonic patterning, emphasizing the necessity of analyzing mutant phenotypes at multiple developmental stages.     

Regenerating islet-derived 1α (Reg-1α) protein is new neuronal secreted factor that stimulates neurite outgrowth via exostosin Tumor-like 3 (EXTL3) receptor

Regenerating islet-derived 1α (Reg-1α)/lithostathine, a member of a family of secreted proteins containing a C-type lectin domain, is expressed in various organs and plays a role in proliferation, differentiation, inflammation, and carcinogenesis of cells of the digestive system. We previously reported that Reg-1α is overexpressed during the very early stages of Alzheimer disease, and Reg-1α deposits were detected in the brain of patients with Alzheimer disease. However, the physiological function of Reg-1α in neural cells remains unknown. Here, we show that Reg-1α is expressed in neuronal cell lines (PC12 and Neuro-2a) and in rat primary hippocampal neurons (E17.5). Reg-1α is mainly localized around the nucleus and at the membrane of cell bodies and neurites. Transient overexpression of Reg-1α or addition of recombinant Reg-1α significantly increases the number of cells with longer neurites by stimulating neurite outgrowth. These effects are abolished upon down-regulation of Reg-1α by siRNA and following inhibition of secreted Reg-1α by antibodies. Moreover, Reg-1α colocalizes with exostosin tumor-like 3 (EXTL3), its putative receptor, at the membrane of these cells. Overexpression of EXTL3 increases the effect of recombinant Reg-1α on neurite outgrowth, and Reg-1α is not effective when EXTL3 overexpression is down-regulated by shRNA. Our findings indicate that Reg-1α regulates neurite outgrowth and suggest that this effect is mediated by its receptor EXTL3.     

Heparan sulfate 3-O-sulfotransferase 2 (HS3ST2) displays an unexpected subcellular localization in the plasma membrane

Background

Heparan sulfate (HS) 3-O-sulfation can be catalysed by seven 3-O-sulfotransferases (HS3STs) in humans, still it is the rarest modification in HS and its biological function is yet misunderstood. HS3ST2 and HS3ST3B exhibit the same activity in vitro. They are however differently expressed in macrophages depending on cell environment, which suggests that they may be involved in distinct cellular processes. Here, we hypothesized that both isozymes might also display distinct subcellular localizations.