SummaryTissue inhibitor of metalloproteinase 3 (TIMP-3) is an important regulator of extracellular matrix (ECM) turnover. TIMP-3 binds to sulfated ECM glycosaminoglycans or is endocytosed by cells via low-density lipoprotein receptor-related protein 1 (LRP-1). Here, we report that heparan sulfate (HS) and chondroitin sulfate E (CSE) selectively regulate postsecretory trafficking of TIMP-3 by inhibiting its binding to LRP-1. HS and CSE also increased TIMP-3 affinity for glycan-binding metalloproteinases, such as adamalysin-like metalloproteinase with thrombospondin motifs 5 (ADAMTS-5), by reducing the dissociation rate constants. The sulfation pattern was crucial for these activities because monosulfated or truncated heparin had a reduced ability to bind to TIMP-3 and increase its affinity for ADAMTS-5. Therefore, sulfation of ECM glycans regulates the levels and inhibitory activity of TIMP-3 and modulates ECM turnover, and small mimicries of sulfated glycans may protect the tissue from the excess destruction seen in diseases such as osteoarthritis, cancer, and atherosclerosis.

Hereditary multiple exostoses (HME) is an autosomal dominant skeletal disorder with wide variation in clinical phenotype and is caused by heterogeneous germline mutations in two of the Ext genes, EXT-1 and EXT-2, which encode ubiquitously expressed glycosyltransferases involved in the polymerization of heparan sulfate (HS) chains. To examine whether the Ext mutation could affect HS structures and amounts in HME patients being heterozygous for the Ext genes, we collected blood from patients and healthy individuals, separated it into plasma and cellular fractions and then isolated glycosaminoglycans (GAGs) from those fractions. A newly established method consisting of a combination of selective ethanol precipitation of GAGs, digestion of GAGs recovered on the filter-cup by direct addition of heparitinase or chondroitinase reaction solution and subsequent high-performance liquid chromatography of the unsaturated disaccharide products enabled the analysis using the least amount of blood (200 µL). We found that HS structures of HME patients were almost similar to those of controls in both plasma and cellular fractions. However, interestingly, although both the amounts of HS and chondroitin sulfate (CS) varied depending on the different individuals, the amounts of HS in both the plasma and cellular fractions of HME patient samples were decreased and the ratios of HS to CS (HS/CS) of HME patient samples were almost half those of healthy individuals. The results suggest that HME patients' blood exhibited reduced HS amounts and HS/CS ratios, which could be used as a diagnostic biomarker for HME.

Background: Heparin regulates mast cell proteases. Results: Both HS6ST-1 and HS6ST-2 are involved in 6-O-sulfation of heparin. The contents of tryptase and CPA in mast cells depend on 6-O-sulfation of heparin but chymase does not. Conclusion:The 6-O-sulfation pattern regulates differently the storage of MC-specific proteases. Significance: The fine structure of heparin may be essential for MC homeostasis.

Glycosaminoglycans (GAGs) are linear polysaccharides having disaccharide building blocks consisting of an amino sugar (N-acetylglucosamine, or N-acetylgalactosamine) and a uronic acid (glucuronic acid or iduronic acid) or galactose. Glycosaminoglycans have sulfated residues at various positions except for hyaluronan, and those sulfated residues regulate the biological functions of a wide variety of proteins, primarily through high-affinity interactions mediated by specific patterns/densities of sulfation and sugar sequences. Alteration of GAG structure is associated with a number of disease conditions and therefore the analyses of GAG structures and their sulfation patterns are important for the development of disease biomarkers and for treatment options. Extensive structural and quantitative analyses of GAGs from human blood are largely unexplored which may be due to the exhaustive isolation process because of the presence of too much interfering proteins and lipids such as serum albumin. Therefore we established a new GAG isolation method using the least amount (~200 μl) of human blood, consisting of a combination of proteolytic digestion and selective ethanol precipitation of GAGs, digestion of GAGs recovered on the filter cup by direct addition of GAG lyase reaction solution, and subsequent high-pressure liquid chromatography of unsaturated disaccharide products that enable to analyze GAG structures and contents. This isolation method offers an 80 % recovery of GAGs and can be applied to analyze a minute GAG content (≥1 nmol) from the least amount of biological fluids. Hence the method could be useful for the development of disease biomarkers.