Systemic Glycosaminoglycan Clearance by HARE/Stabilin-2 Activates Intracellular Signaling.

Scavenger receptors perform essential functions, critical to maintaining mammalian physiologic homeostasis by continuously clearing vast numbers of biomolecules from blood, interstitial fluid and lymph. Stabilin-2 (Stab2) and the Hyaluronic Acid Receptor for Endocytosis (HARE), a proteolytic isoform of Stab2, are important scavenger receptors responsible for the specific binding and internalization (leading to degradation) of 22 discrete molecules, macromolecular complexes and cell types. One-third of these ligands are glycosaminoglycans (GAGs). Full-length Stab2, but not HARE, mediates efficient phagocytosis of apoptotic cells and bacteria via binding to target surface ligands. HARE, the C-terminal half of Stab2, mediates endocytosis of all the known soluble ligands. HA was the first ligand identified, in 1981, prior to receptor purification or cloning. Seven other GAG ligands were subsequently identified: heparin, dermatan sulfate, chondroitin and chondroitin sulfates A, C, D and E. Synthetic dextran sulfate is also a GAG mimic and ligand. HARE signaling during HA endocytosis was first discovered in 2008, and we now know that activation of HARE/Stab2 signaling is stimulated by receptor-mediated endocytosis or phagocytosis of many, but not all, of its ligands. This review focuses on the HARE-mediated GAG activation of intracellular signaling, particularly the Extracellular Signal-Regulated Kinase 1/2 pathway.

A TLR/AKT/FoxO3 immune tolerance-like pathway disrupts the repair capacity of oligodendrocyte progenitors.

Cerebral white matter injury (WMI) persistently disrupts myelin regeneration by oligodendrocyte progenitor cells (OPCs). We identified a specific bioactive hyaluronan fragment (bHAf) that downregulates myelin gene expression and chronically blocks OPC maturation and myelination via a tolerance-like mechanism that dysregulates pro-myelination signaling via AKT. Desensitization of AKT occurs via TLR4 but not TLR2 or CD44. OPC differentiation was selectively blocked by bHAf in a maturation-dependent fashion at the late OPC (preOL) stage by a noncanonical TLR4/TRIF pathway that induced persistent activation of the FoxO3 transcription factor downstream of AKT. Activated FoxO3 selectively localized to oligodendrocyte lineage cells in white matter lesions from human preterm neonates and adults with multiple sclerosis. FoxO3 constraint of OPC maturation was bHAf dependent, and involved interactions at the FoxO3 and MBP promoters with the chromatin remodeling factor Brg1 and the transcription factor Olig2, which regulate OPC differentiation. WMI has adapted an immune tolerance-like mechanism whereby persistent engagement of TLR4 by bHAf promotes an OPC niche at the expense of myelination by engaging a FoxO3 signaling pathway that chronically constrains OPC differentiation.

Hyaluronan synthase control of synthesis rate and hyaluronan product size are independent functions differentially affected by mutations in a conserved tandem B-X7-B motif.

Hyaluronan synthases (HAS) normally make large (>MDa) hyaluronan (HA) products. Smaller HA fragments (e.g. 100-400 kDa) produced in vivo are associated with inflammation and cell signaling by HA receptors that bind small, but not large, HA. Although HA fragments can arise from breakdown by hyaluronidases, HAS might also be regulated directly to synthesize small HA. Here we examined the Streptococcus equisimilis HAS (SeHAS) C-terminus, which contains a tandem B-X7-B motif (K398-X7-R406-X7-K414), by testing the effects of 27 site-specific scanning mutations and 7 C-terminal truncations on HA synthesis activity and weight-average mass. Although HAS enzymes cannot be HA-binding proteins, these motifs are highly conserved within the Class I HAS family. Fifteen Arg406 mutants made large MDa HA (86-110% wildtype size), with specific activities from 70% to 177% of wildtype. In contrast, 10 of 12 Lys398 mutants made HA that was 8-14% of wildtype size (≤250-480 kDa), with specific activities from 14% to 64% of wildtype. Four nearly inactive (2% wildtype activity) C-terminal truncation mutants made MDa HA (56-71% wildtype). The results confirm earlier findings with Cys-mutants [Weigel PH, Baggenstoss BA. 2012. Hyaluronan synthase polymerizing activity and control of product size are discrete enzyme functions that can be uncoupled by mutagenesis of conserved cysteines. Glycobiology 22:1302-1310] that HAS uses two independent activities to control HA size and HA synthesis rate; these are two separate functions. We conclude that HAS regulatory modifications that alter tandem B-X7-B motif conformation could mimic these mutagenesis-induced effects, allowing HAS in vivo to make small HA directly. The results also support a model in which the tandem-motif region is part of the intra-HAS pore and interacts directly with HA.

Hyaluronic acid receptor for endocytosis (HARE)-mediated endocytosis of hyaluronan, heparin, dermatan sulfate, and acetylated low density lipoprotein (AcLDL), but not chondroitin sulfate types A, C, D, or E, activates NF-κB-regulated gene expression.

The hyaluronan (HA) receptor for endocytosis (HARE; Stab2) clears 14 systemic ligands, including HA and heparin. Here, we used NF-κB promoter-driven luciferase reporter assays to test HARE-mediated intracellular signaling during the uptake of eight ligands, whose binding sites in the HARE ectodomain were mapped by competition studies (Harris, E. N., and Weigel, P. H. (2008) Glycobiology 18, 638-648). Unique intermediate size Select-HA(TM), heparin, dermatan sulfate, and acetylated LDL stimulated dose-dependent HARE-mediated NF-κB activation of luciferase expression, with half-maximal values of 10-25 nM. In contrast, chondroitin sulfate types A, C, D, and E did not stimulate NF-κB activation. Moreover, degradation of endogenous IkB-α (an NF-κB inhibitor) was stimulated only by the signaling ligands. The stimulatory activities of pairwise combinations of the four signaling ligands were additive. The four nonstimulatory chondroitin sulfate types, which compete for HA binding, also effectively blocked HA-stimulated signaling. Clathrin siRNA decreased clathrin expression by ∼50% and completely eliminated NF-κB-mediated signaling by all four ligands, indicating that activation of signaling complexes occurs after endocytosis. These results indicate that HARE not only binds and clears extracellular matrix degradation products (e.g. released normally or during infection, injury, tumorigenesis, or other stress situations) but that a subset of ligands also serves as signaling indicator ligands. HARE may be part of a systemic tissue-stress sensor feedback system that responds to abnormal tissue turnover or damage as a danger signal; the signaling indicator ligands would reflect the homeostatic status, whether normal or pathological, of tissue cells and biomatrix components.

Methods for measuring Class I membrane-bound hyaluronan synthase activity.

Detecting and quantifying hyaluronan (HA) made by Class I HA synthase (HAS) and determining the level of activity of these membrane-bound enzymes is critical in studies to understand the normal biology of HA and how changes in HAS activity and HA levels or size are important in inflammatory and other diseases, tumorigenesis, and metastasis. Unlike the products made by the vast majority of glycosyltransferases, HA products are more complicated since they are made as a heterogeneous population of sizes spanning a broad mass range. Three radioactive and nonradioactive assay methods are described that can give the amount of HA made with or without information about the distribution of product sizes.

Tumor necrosis factor-stimulated gene-6 (TSG-6) amplifies hyaluronan synthesis by airway smooth muscle cells.

We tested the hypothesis that the artificial addition of heavy chains from inter-α-inhibitor to hyaluronan (HA), by adding recombinant TSG-6 (TNF-stimulated gene-6) to the culture medium of murine airway smooth muscle (MASM) cells, would enhance leukocyte binding to HA cables produced in response to poly(I:C). As predicted, the addition of heavy chains to HA cables enhanced leukocyte adhesion to these cables, but it also had several unexpected effects. (i) It produced thicker, more pronounced HA cables. (ii) It increased the accumulation of HA in the cell-associated matrix. (iii) It decreased the amount of HA in the conditioned medium. Importantly, these effects were observed only when TSG-6 was administered in the presence of poly(I:C), and TSG-6 did not exert any effect on its own. Increased HA synthesis occurred during active, poly(I:C)-induced HA synthesis and did not occur when TSG-6 was added after poly(I:C)-induced HA synthesis was complete. MASM cells derived from TSG-6(-/-), HAS1/3(-/-), and CD44(-/-) mice amplified HA synthesis in response to poly(I:C) + TSG-6 in a manner similar to WT MASM cells, demonstrating that they are expendable in this process. We conclude that TSG-6 increases the accumulation of HA in the cell-associated matrix, partially by preventing its dissolution from the cell-associated matrix into the conditioned medium, but primarily by inducing HA synthesis.

Hyaluronan turnover and hypoxic brown adipocytic differentiation are co-localized with ossification in calcified human aortic valves.

The calcification process in aortic stenosis has garnered considerable interest but only limited investigation into selected signaling pathways. This study investigated mechanisms related to hypoxia, hyaluronan homeostasis, brown adipocytic differentiation, and ossification within calcified valves. Surgically explanted calcified aortic valves (n=14) were immunostained for markers relevant to these mechanisms and evaluated in the center (NodCtr) and edge (NodEdge) of the calcified nodule (NodCtr), tissue directly surrounding nodule (NodSurr); center and tissue surrounding small "prenodules" (PreNod, PreNodSurr); and normal fibrosa layer (CollFibr). Pearson correlations were determined between staining intensities of markers within regions. Ossification markers primarily localized to NodCtr and NodEdge, along with markers related to hyaluronan turnover and hypoxia. Markers of brown adipocytic differentiation were frequently co-localized with markers of hypoxia. In NodCtr and NodSurr, brown fat and ossification markers correlated with hyaluronidase-1, whereas these markers, as well as hypoxia, correlated with hyaluronan synthases in NodEdge. The protein product of tumor necrosis factor-α stimulated gene-6 strongly correlated with ossification markers and hyaluronidase in the regions surrounding the nodules (NodSurr, PreNodSurr). In conclusion, this study suggests roles for hyaluronan homeostasis and the promotion of hypoxia by cells demonstrating brown fat markers in calcific aortic valve disease.

Hyaluronan synthase polymerizing activity and control of product size are discrete enzyme functions that can be uncoupled by mutagenesis of conserved cysteines.

Streptococcus equisimilis hyaluronan (HA) synthase (SeHAS) contains four cysteines (C226, C262, C281 and C367) that are conserved in the mammalian HAS family. Previous studies of single Cys-to-Ser and all possible Cys-to-Ala mutants of SeHAS found that: the Cys-null mutant is active, Cys modification inhibits HAS activity and the conserved cysteines are clustered at the membrane-enzyme interface in substrate-binding sites (Kumari K, Weigel PH. 2005. Identification of a membrane-localized cysteine cluster near the substrate binding sites of the Streptococcus equisimilis hyaluronan synthase. Glycobiology. 15:529-539). We re-examined these Cys mutants using a single technique (size exclusion chromatography-multi-angle laser light scattering) that allows simultaneous assays on the same sample for both HA synthesis activity and HA product size. Among 18 mutants compared with wild type, 4 showed no change in either function and 3 showed changes in both (decreased activity and HA size). Only one of the two functions was altered in 11 other mutants, which showed either decreased polymerizing activity or product size. No mutants made larger HA, 8 made smaller HA and 10 showed no change in HA size. Nine mutants showed no change in activity and nine were less active. The mutants fell into four of nine possible groups in terms of changes in HA size or synthesis rate (i.e. none, increased or decreased). Specific Cys residues were associated with each mutant group and the pattern of effects on both functions. Thus, the four conserved Cys residues, individually and in specific combinations, influence the rate of sugar assembly by HAS and HA product size, but their participation in one function is independent of the other.

Hyaluronan synthase mediates dye translocation across liposomal membranes.

BACKGROUND: Hyaluronan (HA) is made at the plasma membrane and secreted into the extracellular medium or matrix by phospolipid-dependent hyaluronan synthase (HAS), which is active as a monomer. Since the mechanism by which HA is translocated across membranes is still unresolved, we assessed the presence of an intraprotein pore within HAS by adding purified Streptococcus equisimilis HAS (SeHAS) to liposomes preloaded with the fluorophore Cascade Blue (CB). RESULTS: CB translocation (efflux) was not observed with mock-purified material from empty vector control E. coli membranes, but was induced by SeHAS, purified from membranes, in a time- and dose-dependent manner. CB efflux was eliminated or greatly reduced when purified SeHAS was first treated under conditions that inhibit enzyme activity: heating, oxidization or cysteine modification with N-ethylmaleimide. Reduced CB efflux also occurred with SeHAS K48E or K48F mutants, in which alteration of K48 within membrane domain 2 causes decreased activity and HA product size. The above results used liposomes containing bovine cardiolipin (BCL). An earlier study testing many synthetic lipids found that the best activating lipid for SeHAS is tetraoleoyl cardiolipin (TO-CL) and that, in contrast, tetramyristoyl cardiolipin (TM-CL) is an inactivating lipid (Weigel et al, J. Biol. Chem. 281, 36542, 2006). Consistent with the effects of these CL species on SeHAS activity, CB efflux was more than 2-fold greater in liposomes made with TO-CL compared to TM-CL. CONCLUSIONS: The results indicate the presence of an intraprotein pore in HAS and support a model in which HA is translocated to the exterior by HAS itself.

Systemic blockade of the hyaluronan receptor for endocytosis prevents lymph node metastasis of prostate cancer.

Tumor progression and metastasis are promoted by the remodeling of organized tissue architecture and engagement of molecular interactions that support tumor cell passage through endothelial barriers. Prostate tumor cells that secrete and turn over excessive quantities of pericellular hyaluronan (HA) exhibit accelerated growth kinetics and spontaneous lymph node metastasis in mice. The HA receptor for endocytosis (HARE) is an endocytic clearance receptor for HA in the liver that is also highly expressed in sinusoidal endothelium of lymph nodes and bone marrow, which are frequent sites of prostate cancer metastasis. In our study, we tested the hypothesis that HARE can act as an endothelial receptor for metastatic tumor cells with pericellular HA. In an orthotopic mouse model of prostate cancer, we delivered a monoclonal antibody against HARE that specifically blocks HA binding and internalization. This treatment fully blocked the formation of metastatic tumors in lymph nodes. No effects on primary tumor growth were observed and the antibody did not induce toxic outcomes in any other tissue. Our results implicate HARE for the first time in potentiation of tumor metastasis and suggest a novel mechanism by which tumor cell-associated HA could promote tissue-specific dissemination. "Published 2012 Wiley Periodicals, Inc. This article is a US Government work, and, as such, is in the public domain in the United States of America."

Clustered Conserved Cysteines in Hyaluronan Synthase Mediate Cooperative Activation by Mg2+ Ions and Severe Inhibitory Effects of Divalent Cations.

Hyaluronan synthase (HAS) uses UDP-GlcUA and UDP-GlcNAc to make hyaluronan (HA). Streptococcus equisimilis HAS (SeHAS) contains four conserved cysteines clustered near the membrane, and requires phospholipids and Mg2+ for activity. Activity of membrane-bound or purified enzyme displayed a sigmoidal saturation profile for Mg2+ with a Hill coefficient of 2. To assess if Cys residues are important for cooperativity we examined the Mg2+ dependence of mutants with various combinations of Cys-to-Ala mutations. All Cys-mutants lost the cooperative response to Mg2+. In the presence of Mg2+, other divalent cations inhibited SeHAS with different potencies (Cu2+~Zn2+ >Co2+ >Ni2+ >Mn2+ >Ba2+ Sr2+ Ca2+). Some divalent metal ions likely inhibit by displacement of Mg2+-UDP-Sugar complexes (e.g. Ca2+, Sr2+ and Ba2+ had apparent Ki values of 2-5 mM). In contrast, Zn2+ and Cu2+ inhibited more potently (apparent Ki ≤ 0.2 mM). Inhibition of Cys-null SeHAS by Cu2+, but not Zn2+, was greatly attenuated compared to wildtype. Double and triple Cys-mutants showed differing sensitivities to Zn2+ or Cu2+. Wildtype SeHAS allowed to make HA prior to exposure to Zn2+ or Cu2+ was protected from inhibition, indicating that access of metal ions to sensitive functional groups was hindered in processively acting HA•HAS complexes. We conclude that clustered Cys residues mediate cooperative interactions with Mg2+ and that transition metal ions inhibit SeHAS very potently by interacting with one or more of these -SH groups.

Rat and human HARE/stabilin-2 are clearance receptors for high- and low-molecular-weight heparins.

The human hyaluronic acid (HA) receptor for endocytosis (HARE/stabilin-2) is the primary clearance receptor for systemic HA, chondroitin sulfates, and heparin, but not for heparan sulfate or keratan sulfate (Harris EN, Weigel JA, Weigel PH. J Biol Chem 283: 17341-17350, 2008). HARE is expressed in the sinusoidal endothelial cells (SECs) of liver and lymph nodes where it acts as a scavenger for uptake and degradation of glycosaminoglycans, both as free chains and proteoglycan fragments. Unfractionated heparin (UFH; approximately 14 kDa) and low-molecular-weight heparin (LMWH; approximately 4 kDa) are commonly used in treatments for thrombosis and cancer and in surgical and dialysis procedures. The reported half-lives of UFH and LMWH in the blood are approximately 1 h and 2-6 h, respectively. In this study, we demonstrate that anti-HARE antibodies specifically block the uptake of LMWH and UFH by isolated rat liver SECs and by human 293 cells expressing recombinant human HARE (hHARE). hHARE has a significant affinity (K(d) = 10 microM) for LMWH, and higher affinity (K(d) = 0.06 microM) for the larger UFH. Rat liver SECs or cells expressing the recombinant 190-kDa HARE isoform internalized both UFH and LMWH, and both heparins cross-compete with each other, suggesting that they share the same binding sites. These cellular results were confirmed in ELISA-like assays using purified soluble 190-hHARE ectodomain. We conclude that both UFH and LMWH are cleared by HARE/Stab2 and that the differences in the affinities of HARE binding to LMWH and UFH likely explain the longer in vivo circulating half-life of LMWH compared with UFH.

Abundance and location of proteoglycans and hyaluronan within normal and myxomatous mitral valves.

INTRODUCTION: Extracellular matrix changes occur in many heart valve pathologies. For example, myxomatous mitral valves are reported to contain excess proteoglycans and hyaluronan. However, it is unknown which specific proteoglycans are altered in myxomatous valves. Because proteoglycans perform varied functions in connective tissues, this study was designed to identify and localize three matrix-associated proteoglycans, as well as hyaluronan and the hyaluronan receptor for endocytosis, within myxomatous and normal mitral valves. METHODS: Human mitral posterior leaflets (control, n=6-9; myxomatous, n=14-21; mean age, 61 years for all groups) were histochemically stained for proteoglycan core proteins, hyaluronan, and the hyaluronan receptor for endocytosis. Stain intensity was semiquantitatively graded to determine differences in marker abundance between normal and myxomatous valves. The proteoglycans were localized to different regions of the leaflet by correspondence to parallel Movat-stained sections. RESULTS: The proteoglycans decorin, biglycan, and versican were more abundant in myxomatous valves than in normal controls (P<.03). There was a gender effect on proteoglycan presence, but no age-related trends were observed. Hyaluronan and the hyaluronan receptor for endocytosis were distributed throughout all valves. There was no significant difference in hyaluronan between groups, but expression of the hyaluronan receptor for endocytosis was reduced in myxomatous valves compared to normal controls (P<.002). CONCLUSION: Excess decorin, biglycan, and versican may be associated with the remodeling of other matrix components in myxomatous mitral valves. Decreased expression of the hyaluronan receptor for endocytosis in myxomatous valves suggests that hyaluronan metabolism could be altered in myxomatous mitral valve disease. These findings contribute towards elucidating the pathogenesis of myxomatous mitral valve disease and developing potential new therapies.

The human hyaluronan receptor for endocytosis (HARE/Stabilin-2) is a systemic clearance receptor for heparin.

The hyaluronic acid receptor for endocytosis (HARE; also designated Stabilin-2) mediates systemic clearance of hyaluronan and chondroitin sulfates from the vascular and lymphatic circulations. The internalized glycosaminoglycans are degraded in lysosomes, thus completing their normal turnover process. Sinusoidal endothelial cells of human liver, lymph node, and spleen express two HARE isoforms of 315 and 190 kDa. Here we report that the 190- and 315-kDa HARE isoforms, expressed stably either in Flp-In 293 cell lines or as soluble ectodomains, specifically bind heparin (Hep). The K(d) for Hep binding to purified 190- and 315-kDa HARE ectodomains was 17.2 +/- 4.9 and 23.4 +/- 5.3 nm, respectively. Cells expressing HARE readily and specifically internalized (125)I-streptavidin-biotin-Hep complexes, which was inhibited >70% by hyperosmolar conditions, confirming that uptake is mediated by the clathrin-coated pit pathway. Internalization of Hep occurred for many hours with an estimated HARE recycling time of approximately 12 min. Internalized fluorescent streptavidin-biotin-Hep was present in a typical endocytic vesicular pattern and was delivered to lysosomes. We conclude that HARE in the sinusoidal endothelial cells of lymph nodes and liver likely mediates the efficient systemic clearance of Hep and many different Hep-binding protein complexes from the lymphatic and vascular circulations.

Recombinant human hyaluronan synthase 3 is phosphorylated in mammalian cells.

Hyaluronan is a ubiquitous component of vertebrate extracellular and cell-associated matrices that serves as a key structural component of skin, cartilage, eyes and joints, and plays important roles in dynamic cellular processes, including embryogenesis, inflammation, wound healing and metastasis. Hyaluronan is synthesized by three homologous hyaluronan synthases designated HAS1, HAS2 and HAS3 that differ in their tissue distribution, regulation and enzymatic characteristics. Some progress has been made in characterizing regulation of HAS transcripts and in distinguishing the enzymatic properties of the various HAS isoforms, but essentially nothing is known about their possible regulation by posttranslational modification. Using [32P]P(i) radiolabelling of a recombinant FLAG (DYKDDDDK) epitope-tagged version of human HAS3 expressed in COS-7 cells, we show that HAS3 is serine-phosphorylated and that this phosphorylation can be enhanced by a number of effectors--most significantly by a membrane-permeable analogue of cAMP. By employing a novel FLAG-tagged phosphorylated reference protein derived from EGFP (enhanced green fluorescent protein), we were able to estimate the stoichiometry of FLAG-HAS3 phosphorylation. It was approx. 0.11 in unstimulated cells and increased to as much as 0.32 in cells stimulated with 8-(4-chlorophenylthio)-cAMP.

Hyaluronan and its receptors in mucoepidermoid carcinoma.

BACKGROUND: Hyaluronan (HA) is a prominent extracellular matrix component undergoing continuous production and degradation. Increased HA levels have been described in a variety of tumors. The objective of this study was to examine the staining patterns of HA and two of its associated receptors (CD44 and HARE) in relation to the metastatic potential of mucoepidermoid carcinoma (MC). Immunohistochemical staining of preserved surgical specimens was used. METHODS: Tissues from 12 patients with a histologic diagnosis of salivary MC (10 parotid, one submandibular gland, one minor salivary gland) were studied. Half (six of 12) of the patients had regional metastases. Tumor, normal salivary tissue, and regional lymph nodes were stained for HA, CD44, and HARE expression. Specimens were graded for staining intensity and a percent of the specimen stained. RESULTS: Normal salivary tissue did not demonstrate epithelial cell surface HA expression, whereas HA was expressed on tumor cells and in regional lymph nodes containing metastases. These differences were both significant using Student's t test (p < .00002, and p < .0022, respectively). Tumors with positive nodes tended to have greater cell surface HA. Decreased expression or downregulation of HARE was also noted in involved lymph nodes. No differences in CD44 expression were seen between primary specimens and lymph nodes. The observed staining patterns for CD44 and HARE were not reflective of the metastatic potential of the primary MC. CONCLUSIONS: Increased HA expression was seen on mucoepidermoid carcinoma cells compared with adjacent normal salivary gland epithelium. This observation may assist in explaining the development of regional metastasis in these tumors. We did not identify specific HA, CD44, or HARE staining patterns in primary lesions that were predictive of regional metastases.

Identification of a membrane-localized cysteine cluster near the substrate-binding sites of the Streptococcus equisimilis hyaluronan synthase.

The membrane-bound hyaluronan synthase (HAS) from Streptococcus equisimilis (seHAS), which is the smallest Class I HAS, has four cysteine residues (positions 226, 262, 281, and 367) that are generally conserved within this family. Although Cys-null seHAS is still active, chemical modification of cysteine residues causes inhibition of wild-type enzyme. Here we studied the effects of N-ethylmaleimide (NEM) treatment on a panel of seHAS Cys-mutants to examine the structural and functional roles of the four cysteine residues in the activity of the enzyme. We found that Cys226, Cys262, and Cys281 are reactive with NEM, but Cys367 is not. Substrate protection studies of wild-type seHAS and a variety of Cys-mutants revealed that binding of UDP-GlcUA, UDP-GlcNAc, or UDP can protect Cys226 and Cys262 from NEM inhibition. Inhibition of the six double Cys-mutants of seHAS by sodium arsenite, which can cross-link vicinyl sulfhydryl groups, also supported the conclusion that Cys262 and Cys281 are close enough to be cross-linked. Similar results indicated that Cys281 and Cys367 are also very close in the active enzyme. We conclude that three of the four Cys residues in seHAS (Cys262, Cys281, and Cys367) are clustered very close together, that these Cys residues and Cys226 are located at the inner surface of the cell membrane, and that Cys226 and Cys262 are located in or near a UDP binding site.

Injured brain endothelial cells release neurotoxic thrombin.

The multifunctional serine protease thrombin has been shown to be neurotoxic in vitro and in vivo and is demonstrable in the Alzheimer disease (AD) brain. We have documented that in AD the cerebral microvasculature is a source of inflammatory and neurotoxic proteins. The objective of this study was to determine if injured brain endothelial cells could be a source of neurotoxic thrombin. Brain endothelial cells were incubated with either sodium nitroprusside (SNP, 10 microM), inflammatory proteins (IL-1beta, IL-6, TNFalpha, LPS, IFNgamma) or the PKC inhibitor bisindolymaleimide (1 microM) for 24 h and conditioned media collected. Endothelial cell conditioned medium was incubated with purified apolipoprotein E4 (apoE4) for 24 h, and then analyzed for neurotoxic activity against primary cortical cultures and for apoE4 fragments by western blot. Endothelial cell conditioned medium collected after treatment with either SNP, inflammatory proteins, or the PKC inhibitor bisindolymaleimide, demonstrated a significant (p < 0.005) level of thrombin activity, the presence of apoE4 fragments, and was capable of evoking neuronal cell death. These data demonstrate that endothelial cell injury results in thrombin release and suggest that the brain microcirculation could be a source of neurotoxic factors in AD.

The position of cysteine relative to the transmembrane domain is critical for palmitoylation of H1, the major subunit of the human asialoglycoprotein receptor.

The mammalian hepatic asialoglycoprotein receptor (ASGP-R) is an endocytic recycling receptor that mediates the internalization of desialylated glycoproteins and their delivery to lysosomes where they are degraded. The human ASGP-R is a hetero-oligomeric complex composed of two subunits designated H1 and H2. Both subunits are palmitoylated at the cytoplasmic Cys residues near their transmembrane domains (TMD). The cytoplasmic Cys(36) in H1 is located at a position that is five amino acids from the transmembrane junction. Because the sequences of subunits in all mammalian ASGP-R species are highly conserved especially at the region near the palmitoylated Cys, we sought to identify a recognition signal for the palmitoylation of H1. Various types of H1 mutants were created by site-directed or deletion mutagenesis including alteration of the amino acids surrounding Cys(36), replacing portions of the TMD with that of a different protein and partial deletion of the cytoplasmic domain as well as transposing the palmitoylated Cys to positions further away from the TMD. Mutant H1 cDNAs were transiently expressed in COS-7 cells, and the H1 proteins were analyzed after metabolic labeling with [(3)H]palmitate. The results indicate that neither the native amino acid sequence surrounding Cys(36) nor the majority of the cytoplasmic domain sequence is critical for palmitoylation. Palmitoylation was also not dependent on the native TMD of H1. In contrast, the attachment of palmitate was abolished if the Cys residue was transposed to a position that was 30 amino acids away from the transmembrane border. We conclude that the spacing of a Cys residue relative to the TMD in the primary protein sequence of H1 is the major determinant for successful palmitoylation.

Palmitoylation-defective asialoglycoprotein receptors are normal in their cellular distribution and ability to bind ligand, but are defective in ligand uptake and degradation.

The hepatic asialoglycoprotein receptor (ASGP-R) is an endocytic recycling receptor that mediates the endocytosis of desialylated glycoproteins. The human ASGP-R is composed of two homologous subunits, H1 and H2, and the cytoplasmic Cys residues in both subunits are palmitoylated. To study the effects of palmitoylation on ASGP-R activity and function, we generated four types of stably transfected cell lines in SK-Hep-1 hepatoma cells, expressing wild-type, or partially or completely palmitoylation-defective ASGP-Rs containing Cys-to-Ser mutations in either one or both subunits. Scatchard analysis showed that all four stable cell lines expressed a similar number of binding sites for asialo-orosomucoid, with comparable dissociation constants of approximately 1-3nM. Immunofluorescence confocal microscopy indicated a normal distribution of the palmitoylation-defective H1 and H2 subunits compared to the wild-type. However, cell lines expressing palmitoylation-defective ASGP-Rs had markedly reduced rates of ligand uptake and degradation compared to cells expressing wild-type ASGP-Rs. We conclude that failure to palmitoylate Cys residues in either or both subunits of human ASGP-Rs results in very inefficient uptake and degradation of ligands.