TMEM165 a new player in proteoglycan synthesis: loss of TMEM165 impairs elongation of chondroitin- and heparan-sulfate glycosaminoglycan chains of proteoglycans and triggers early chondrocyte differentiation and hypertrophy.

TMEM165 deficiency leads to skeletal disorder characterized by major skeletal dysplasia and pronounced dwarfism. However, the molecular mechanisms involved have not been fully understood. Here, we uncover that TMEM165 deficiency impairs the synthesis of proteoglycans by producing a blockage in the elongation of chondroitin-and heparan-sulfate glycosaminoglycan chains leading to the synthesis of proteoglycans with shorter glycosaminoglycan chains. We demonstrated that the blockage in elongation of glycosaminoglycan chains is not due to defect in the Golgi elongating enzymes but rather to availability of the co-factor Mn2+. Supplementation of cell with Mn2+ rescue the elongation process, confirming a role of TMEM165 in Mn2+ Golgi homeostasis. Additionally, we showed that TMEM165 deficiency functionally impairs TGFß and BMP signaling pathways in chondrocytes and in fibroblast cells of TMEM165 deficient patients. Finally, we found that loss of TMEM165 impairs chondrogenic differentiation by accelerating the timing of Ihh expression and promoting early chondrocyte maturation and hypertrophy. Collectively, our results indicate that TMEM165 plays an important role in proteoglycan synthesis and underline the critical role of glycosaminoglycan chains structure in the regulation of chondrogenesis. Our data also suggest that Mn2+ supplementation may be a promising therapeutic strategy in the treatment of TMEM165 deficient patients.

Novel glycosaminoglycan precursors as anti-amyloid agents, part III.

In vivo amyloids consist of two classes of constituents. The first is the disease-defining protein, e.g., amyloid beta (Abeta) in Alzheimer's disease (AD). The second is a set of common structural components that usually are the building blocks of basement membrane (BM), a tissue structure that serves as a scaffold onto which cells normally adhere. In vitro binding interactions between one of these BM components and amyloidogenic proteins rapidly change the conformation of the amyloidogenic protein into amyloid fibrils. The offending BM component is a heparan sulfate (HS) proteoglycan (HSPG), part of which is protein, and the remainder is a specific linear polysaccharide that is the portion responsible for binding and imparting the typical amyloid structure to the amyloid precursor protein/peptide. Our past work has demonstrated that agents that inhibit the binding between HS and the amyloid precursor are effective antiamyloid compounds both in vitro and in vivo. Similarly, 4-deoxy analogs of glucosamine (a precursor of HS biosynthesis) are effective antiamyloid compounds both in culture and in vivo. Our continuing work concerns (1) the testing of our 4-deoxy compounds in a mouse transgenic model of AD, and (2) the continuing design and synthesis of modified sugar precursors of HS, which when incorporated into the polysaccharide will alter its structure so that it loses its amyloid-inducing properties. Since our previous report, 14 additional compounds have been designed and synthesized based on the known steps involved in HS biosynthesis. Of these, eight have been assessed for their effect on HS biosynthesis in hepatocyte tissue cultures, and the two anomers of a 4-deoxy-D-glucosamine analog have been assessed for their inflammation-associated amyloid (AA amyloid) inhibitory properties in vivo. The promising in vivo results with these two compounds have prompted studies using a murine transgenic model of brain Abeta amyloidogenesis. A macrophage tissue-culture model of AA amyloidogenesis has been devised based on the work of Kluve-Beckerman et al. and modified so as to assess compounds in the absence of potential in vivo confounding variables. Preliminary results indicate that the anomers of interest also inhibit AA amyloid deposition in macrophage tissue culture. Finally, an in vitro technique, using liver Golgi (the site of HS synthesis) rather than whole cells, has been devised to directly assess the effect of analogs on HS biosynthesis. The majority of the novel sugars prepared to date are analogs of N-acetylglucosamine. They have been modified either at the 2-N, C-3, C-4, or C-3 and C-4 positions. Results with the majority of the 2-N analogs suggest that hepacyte N-demethylases remove the N-substituent removal. Several of these have the desired effect on HS biosynthesis using hepatocyte cultures and will be assessed in the culture and in vivo AA amyloid models. To date 3-deoxy and 3,4-dideoxy analogs have failed to affect HS synthesis significantly. Compounds incorporating the 6-deoxy structural feature are currently being designed and synthesized.

Heparan sulfate chains with antimitogenic properties arise from mesangial cell-surface proteoglycans.

Heparan sulfate (HS) chains accumulate in both the medium and the cell layer of mesangial cell cultures. When given in fresh medium to quiescent cultures at naturally occurring concentrations, they suppress entry into the cell cycle and progression to DNA synthesis. We have attempted to identify the proteoglycan (PG) source of the antimitogenic HS chains from mesangial cell layers (HS(c)) and medium (HS(c)). When cells were labeled for 16 hours with [35S]sulfate, 25% of the label was found in intracellular HS chains and 5% in extracellular HSPGs. Cell-surface HSPGs accounted for the remaining 70% of the label associated with cell-layer HS and were released by either trypsin or 2% Triton X-100. About 20% of this cell-surface fraction was released by treatment with phosphatidylinositol-specific phospholipase C (PI-PLC), and probably represents glypican-like PG; glypican mRNA was present in the cells. The remainder of this fraction could be incorporated into liposomes, indicating the presence of hydrophobic transmembrane regions suggestive of syndecans. Upon purification and deglycosylation, an antiserum to rat liver HSPGs that reacts primarily with syndecan-2 showed a strong signal corresponding to this protein and three weaker bands that may represent additional syndecans. mRNAs for syndecan-1, -2, and -4 were present in the cultures. Syndecan-1 and -2 mRNAs were increased 30 minutes after stimulation of quiescent rat mesangial cells (RMCs) with serum. Heparin, HS(c), and HS(m) all prevented this increase. Syndecan-4 mRNA was not affected by serum, heparin, or HS. In pulse-chase experiments, the amount of 35S appearing in the cellular protein-free HS fraction was accounted for almost entirely by cell-surface PGs, as matrix-associated label was a minor contribution at the end of the pulse-labeling. The appearance of [35S]HS in cell extracts was unaffected by phospholipase C treatment, indicating that turnover of the newly labeled syndecan fraction is the source of the antimitogenic HS chains.