Mechanisms of cell death in primary cortical neurons and PC12 cells.

Increasing evidence suggests that the regulation of neuronal cell death is complex. In this study we compared the neurotoxic effects of tumor necrosis factor-alpha (TNFalpha), nitric oxide, and thrombin on primary rat cortical cell cultures and the neuronal PC12 cell line. Release of lactate dehydrogenase (LDH) and the intracellular accumulation of nucleosomes were used as indicators of necrosis and apoptosis, respectively. There was significant LDH release in both neuronal cell types, however, the pattern of LDH release was variable and agonist-dependent. In response to the nitric oxide generator, sodium nitroprusside (SNP), cortical cells exhibited pronounced LDH release and dramatic morphologic changes, whereas in differentiated PC12 cells, TNFalpha evoked release of LDH with no associated morphologic changes. Both neuronal cell types, but not undifferentiated PC12 cells, responded to TNFalpha and thrombin with increased apoptosis. Caspase inhibition, but not antioxidant treatment, reduced nucleosome accumulation in primary cortical cells, but not in differentiated PC12 cells. In the differentiated PC12 cells, caspase inhibition reduced TNFalpha-mediated LDH release, but not nucleosome accumulation. These data suggest mechanisms involved in neuronal cell death utilize multiple pathways that vary depending on the neurotoxic insult and are also influenced by subtle differences among neuronal cell phenotypes.

Topological organization of the hyaluronan synthase from Streptococcus pyogenes.

Since we first reported (DeAngelis, P. L., Papaconstantinou, J., and Weigel, P. H. (1993) J. Biol. Chem. 268, 19181-19184) the cloning of the hyaluronan (HA) synthase from Streptococcus pyogenes (spHAS), numerous membrane-bound HA synthases have been discovered in both prokaryotes and eukaryotes. The HASs are unique among enzymes studied to date because they mediate 6-7 discrete functions in order to assemble a polysaccharide containing hetero-disaccharide units and simultaneously effect translocation of the growing HA chain through the plasma membrane. To understand how the relatively small spHAS performs these various functions, we investigated the topological organization of the protein utilizing fusion analysis with two reporter enzymes, alkaline phosphatase and beta-galactosidase, as well as several other approaches. From these studies, we conclude that the NH2 terminus and the COOH terminus, as well as the major portion of a large central domain are localized intracellularly. The first two predicted membrane domains were confirmed to be transmembrane domains and give rise to a very small extracellular loop that is inaccessible to proteases. Several regions of the large internal central domain appear to be associated with, but do not traverse, the membrane. Following the central domain, there are two additional transmembrane domains connected by a second small extracellular loop that also is inaccessible to proteases. The COOH-terminal approximately 25% of spHAS also contains a membrane domain that does not traverse the membrane and may contain extensive re-entrant loops or amphipathic helices. Numerous membrane associations of this latter COOH-terminal region and the central domain may be required to create a pore-like structure through which a growing HA chain can be extruded to the cell exterior. Based on the high degree of similarity among Class I HAS family members, these enzymes may have a similar topological organization for their spHAS-related domains.

Site-directed mutation of conserved cysteine residues does not inactivate the Streptococcus pyogenes hyaluronan synthase.

Hyaluronan synthase (HAS), the enzyme responsible for the production of hyaluronic acid (HA), is a well-conserved membrane-bound protein in both prokaryotes and eukaryotes. This enzyme performs at least six discrete functions in producing a heterodisaccharide polymer of several million molecular weight and extruding it from the cell. Among the conserved motifs and domains within the Class I HAS family are four cysteine residues. Cysteines in many proteins are important in establishing and maintaining tertiary structure or in the coordination of catalytic functions. In the present study we utilized a combination of site-directed mutagenesis, chemical labeling, and kinetic analyses to determine the importance of specific Cys residues for catalysis and structure of the HA synthase from Streptococcus pyogenes (spHAS). The enzyme activity of spHAS was partially inhibited by cysteine-reactive chemical reagents such as N-ethylmaleimide. Quantitation of the number of Cys residues modified by these reagents, using MALDI-TOF mass spectrometry, demonstrated that there are no stable disulfide bonds in spHAS. The six Cys residues of spHAS were then mutated, individually and in various combinations, to serine or alanine. The single Cys-mutants were all kinetically similar to the wild-type enzyme in terms of their V(max) and K(m) values for HA synthesis. The Cys-null mutant, in which all Cys residues were mutated to alanine, retained approximately 66% of wild-type activity, demonstrating that despite their high degree of conservation within the HAS family, Cys residues are not absolutely necessary for HA biosynthesis by the spHAS enzyme.

Identification of the hyaluronan receptor for endocytosis (HARE).

Rat liver sinusoidal endothelial cells (LECs) express two hyaluronan (HA) receptors, of 175 and 300 kDa, responsible for the endocytic clearance of HA. We have characterized eight monoclonal antibodies (mAbs) raised against the 175-kDa HA receptor partially purified from rat LECs. These mAbs also cross-react with the 300-kDa HA receptor. The 175-kDa HA receptor is a single protein, whereas the 300-kDa species contains three subunits, alpha, beta, and gamma at 260, 230, and 97 kDa, respectively (Zhou, B., Oka, J. A., and Weigel, P. H. (1999) J. Biol. Chem. 274, 33831-33834). The 97-kDa subunit was not recognized by any of the mAbs in Western blots. Based on their cross-reactivity with these mAbs, the 175-, 230-, and 260-kDa proteins appear to be related. Two of the mAbs inhibit (125)I-HA binding and endocytosis by LECs at 37 degrees C. All of these results confirm that the mAbs recognize the bone fide LEC HA receptor. Indirect immunofluoresence shows high protein expression in liver sinusoids, the venous sinuses of the red pulp in spleen, and the medullary sinuses of lymph nodes. Because the tissue distribution for this endocytic HA receptor is not unique to liver, we propose the name HARE (HA receptor for endocytosis).

Human intestinal epithelial cells express a novel receptor for IgA.

Binding and transport of polymeric Igs (pIgA and IgM) across epithelia is mediated by the polymeric Ig receptor (pIgR), which is expressed on the basolateral surface of secretory epithelial cells. Although an Fc receptor for IgA (FcalphaR) has been identified on myeloid cells and some cultured mesangial cells, the expression of an FcalphaR on epithelial cells has not been described. In this study, binding of IgA to a human epithelial line, HT-29/19A, with features of differentiated colonic epithelial cells, was examined. Radiolabeled monomeric IgA (mIgA) showed a dose-dependent, saturable, and cation-independent binding to confluent monolayers of HT-29/19A cells. Excess of unlabeled mIgA, but not IgG or IgM, competed for the mIgA binding, indicating that the binding was IgA isotype-specific and was not mediated by the pIgR. The lack of competition by asialoorosomucoid and the lack of requirement for divalent cations excluded the possibility that IgA binding to HT-29/19A cells was due to the asialoglycoprotein receptor or beta-1, 4-galactosyltransferase, previously described on HT-29 cells. Moreover, the FcalphaR (CD89) protein and message were undetectable in HT-29/19A cells. FACS analysis of IgA binding demonstrated two discrete populations of HT-29/19 cells, which bound different amounts of mIgA. IgA binding to other colon carcinoma cell lines was also demonstrated by FACS analysis, suggesting that an IgA receptor, distinct from the pIgR, asialoglycoprotein receptor, galactosyltransferase, and CD89 is constitutively expressed on cultured human enterocytes. The function of this novel IgA receptor in mucosal immunity remains to be elucidated.

Cerebrovasculature-mediated neuronal cell death.

The presence of significant vascular disease in patients with Alzheimer's disease (AD) and the recognition of the ApoE genotype as a risk factor for both coronary disease and AD support an association between AD and vascular disease. It is our hypothesis that brain microvessels contribute to the pathogenesis of AD by producing soluble factors that injure or kill neurons. In this study we report that AD microvessels produce factors that are noxious to neurons and that these vessels can evoke neuronal cell death in vitro. In these experiments, microvessels are isolated from the cerebral cortices of AD patients and non-demented elderly and young controls. Microvessels isolated from AD brains produce high levels of a known neurotoxin nitric oxide, compared to vessels from aged-matched controls. In addition, we demonstrate a direct neurotoxic effect of AD microvessels when co-cultured with primary rat cerebral cortical neurons. In contrast, vessels from elderly non-demented donors are less lethal, and brain vessels from younger donors are not neurotoxic. Similarly, AD vessels exhibit a dose-dependent toxicity in co-culture with the human neurons. Finally, treatment of AD microvessels with the protein synthesis inhibitor cycloheximide reduces AD vessel neurotoxicity, suggesting that the neurotoxic factor is a protein. These findings suggest that the cerebral microvasculature is a source of factors that can injure neurons and implicate a novel mechanism of vascular-mediated neuronal cell death in AD.

Purification and subunit characterization of the rat liver endocytic hyaluronan receptor.

The endocytic hyaluronan (HA) receptor of liver sinusoidal endothelial cells (LECs) is responsible for the clearance of HA and other glycosaminoglycans from the circulation in mammals. We report here for the first time the purification of this liver HA receptor. Using lectin and immuno-affinity chromatography, two HA receptor species were purified from detergent-solubilized membranes prepared from purified rat LECs. In nonreducing SDS-polyacrylamide gel electrophoresis (PAGE), these two proteins migrated at 175- and approximately 300 kDa corresponding to the two species previously identified by photoaffinity labeling of live cells as the HA receptor (Yannariello-Brown, J., Frost, S. J., and Weigel, P. H. (1992) J. Biol. Chem. 267, 20451-20456). These two proteins co-purify in a molar ratio of 2:1 (175:300), and both proteins are active, able to bind HA after SDS-PAGE, electrotransfer, and renaturation. After reduction, the 175-kDa protein migrates as a approximately 185-kDa protein and is not able to bind HA. The 300-kDa HA receptor is a complex of three disulfide-bonded subunits that migrate in reducing SDS-PAGE at approximately 260, 230, and 97 kDa. These proteins designated, respectively, the alpha, beta, and gamma subunits are present in a molar ratio of 1:1:1 and are also unable to bind HA when reduced. The 175-kDa protein and all three subunits of the 300-kDa species contain N-linked oligosaccharides, as indicated by increased migration in SDS-PAGE after treatment with N-glycosidase F. Both of the deglycosylated, nonreduced HA receptor proteins still bind HA.

Purification and lipid dependence of the recombinant hyaluronan synthases from Streptococcus pyogenes and Streptococcus equisimilis.

The two hyaluronan synthases (HASs) from Streptococcus pyogenes (spHAS) and Streptococcus equisimilis (seHAS) were expressed in Escherichia coli as recombinant proteins containing His6 tails. Both enzymes were expressed as major membrane proteins, accounting for approximately 5-8% of the total membrane protein. Using nickel chelate affinity chromatography, the HASs were purified to homogeneity from n-dodecyl beta-D-maltoside extracts. High levels of HAS activity could be achieved only if the purified enzymes were supplemented with either bovine or E. coli cardiolipin (CL), although bovine CL gave consistently greater activity. Mass spectroscopic analysis revealed that the fatty acid compositions of these two CL preparations did not overlap. The two HAS enzymes showed similar but distinct activation profiles with the 10 other lipids tested. For example, phosphatidic acid and phosphatidylethanolamine stimulated seHAS, but not spHAS. Phosphatidylserine stimulated both enzymes. spHAS appears to be more CL-specific than seHAS, although both purified enzymes still contain endogenous CL that can not easily be removed. Both seHAS and spHAS were inhibited by phosphatidylcholine, sphingomyelin, and sulfatides and were not substantially stimulated by cerebrosides, phosphatidylglycerol, or phosphatidylinositol. With both HASs, CL increased the Km for UDP-GlcUA, but decreased the Km for UDP-GlcNAc and gave an overall stimulation of Vmax. A kinetic characterization of the two membrane-bound and purified HASs is presented in the accompanying paper (Tlapak-Simmons, V. L., Baggenstoss, B. A., Kumari, K., Heldermon, C., and Weigel, P. H. (1999) J. Biol. Chem. 274, 4246-4253). Both purified HASs became inactive after storage for approximately 5 days at 4 degreesC. Both purified enzymes also lost activity over 4-5 days when stored at -80 degreesC in the presence of CL, but reached a level of activity that then slowly decreased over a period of months. Although the purified enzymes stored in the absence of CL at -80 degreesC were much less active, the enzymes retained this same low level of activity for at least 5 weeks. When both spHAS and seHAS were stored without CL at -80 degreesC, even after 2 months, they could be stimulated by the addition of bovine CL to approximately 60% of the initial activity of the freshly purified enzyme.

Kinetic characterization of the recombinant hyaluronan synthases from Streptococcus pyogenes and Streptococcus equisimilis.

The two hyaluronan synthases (HASs) from Streptococcus pyogenes (spHAS) and Streptococcus equisimilis (seHAS) were expressed in Escherichia coli as recombinant proteins containing His6 tails. The accompanying paper has described the purification and lipid dependence of both HASs, their preference for cardiolipin, and their stability during storage (Tlapak-Simmons, V. L., Baggenstoss, B. A., Clyne, T., and Weigel, P. H. (1999) J. Biol. Chem. 274, 4239-4245). Kinetic characterization of the enzymes in isolated membranes gave Km values for UDP-GlcUA of 40 +/- 4 microM for spHAS and 51 +/- 5 microM for seHAS. In both cases, the Vmax profiles at various concentrations of UDP-GlcNAc were hyperbolic, with no evidence of cooperativity. In contrast, membrane-bound spHAS, but not seHAS, showed sigmoidal behavior as the UDP-GlcNAc concentration was increased, with a Hill number of approximately 2, indicating significant cooperativity. The Hill number for UDP-GlcNAc utilization by seHAS was 1, confirming the lack of cooperativity for UDP-GlcNAc in this enzyme. The Km values for UDP-GlcNAc were 60 +/- 7 microM for seHAS and 149 +/- 3 microM for spHAS in the isolated membranes. The kinetic characteristics of the two affinity-purified HAS enzymes were assessed in the presence of cardiolipin after 8-9 days of storage at -80 degreesC without cardiolipin. With increasing storage time, the enzymes showed a gradual increase in their Km values for both substrates and a decrease in Vmax. Even in the presence of cardiolipin, the detergent-solubilized, purified HASs had substantially higher Km values for both substrates than the membrane-bound enzymes. The KUDP-GlcUA for purified spHAS and seHAS increased 2-4-fold. The KUDP-GlcNAc for spHAS and seHAS increased 4- and 5-fold, respectively. Despite the higher Km values, the Vmax values for the purified HASs were only approximately 50% lower than those for the membrane-bound enzymes. Significantly, purified spHAS displayed the same cooperative interaction with UDP-GlcNAc (nH approximately 2), whereas purified seHAS showed no cooperativity.

Microvessels from Alzheimer's disease brains kill neurons in vitro.

Understanding the pathogenesis of Alzheimer's disease is of widespread interest because it is an increasingly prevalent disorder that is progressive, fatal, and currently untreatable. The dementia of Alzheimer's disease is caused by neuronal cell death. We demonstrate for the first time that blood vessels isolated from the brains of Alzheimer's disease patients can directly kill neurons in vitro. Either direct co-culture of Alzheimer's disease microvessels with neurons or incubation of cultured neurons with conditioned medium from microvessels results in neuronal cell death. In contrast, vessels from elderly nondemented donors are significantly (P<0.001) less lethal and brain vessels from younger donors are not neurotoxic. Neuronal killing by either direct co-culture with Alzheimer's disease microvessels or conditioned medium is dose- and time-dependent. Neuronal death can occur by either apoptotic or necrotic mechanisms. The microvessel factor is neurospecific, killing primary cortical neurons, cerebellar granule neurons, and differentiated PC-12 cells, but not non-neuronal cell types or undifferentiated PC-12 cells. Appearance of the neurotoxic factor is decreased by blocking microvessel protein synthesis with cycloheximide. The neurotoxic factor is soluble and likely a protein, because its activity is heat labile and trypsin sensitive. These findings implicate a novel mechanism of vascular-mediated neuronal cell death in Alzheimer's disease.

Expression and regulation of leukotriene-synthesis enzymes in rat liver cells.

The liver plays a major role in metabolism and elimination of leukotrienes (LT). It produces cysteinyl leukotrienes (cLT), and cLT have been implicated in hepatocellular toxicity in several models of lipopolysaccharide (LPS)-associated liver injury. However, the liver cell types responsible for cLT production are poorly defined, and the expression of the LT-synthesis enzymes, 5-lipoxygenase (5-LO) and LTC4 synthase (LTC4-S), in liver cells has never been demonstrated. The aim of the present study was to examine the ability of rat liver cells to produce cLT by determining whether hepatocytes, Kupffer cells, and sinusoidal endothelial cells express mRNA and enzyme activities of the LT-synthesis enzymes and whether expression is altered by LPS. 5-LO mRNA was expressed in whole liver, and expression was enhanced by LPS. Cell fractionation studies demonstrated that expression was present in Kupffer cells and sinusoidal endothelial cells, but not in hepatocytes. LTC4-S mRNA was detected in whole liver, hepatocytes, and sinusoidal endothelial cells, but not in Kupffer cells. Semiquantitative reverse-transcriptase polymerase chain reaction (RT-PCR) showed that LPS increased LTC4-S expression in hepatocytes by a factor of 3 (n = 3; P < .03). LTC4-S enzyme activity in the microsomal fraction of hepatocytes was also increased from 0.52 +/- 0.13 to 1.90 +/- 0.66 nmol . mg protein-1 . 5 min-1 (n = 6; P < .015) after LPS treatment. These results indicate that hepatocytes do not possess the ability for de novo synthesis of cLT from arachidonic acid, but they may actively participate in cLT production by conjugation of LTA4 with glutathione to produce LTC4. LPS enhances LTC4-S expression in hepatocytes. This intrinsic cLT production may contribute to hepatocellular injury during inflammation.

The active streptococcal hyaluronan synthases (HASs) contain a single HAS monomer and multiple cardiolipin molecules.

The functional sizes of the two streptococcal hyaluronan synthases (HASs) were determined by radiation inactivation analysis of isolated membranes. The native enzymes in membranes from Group A Streptococcus pyogenes HAS and Group C Streptococcus equisimilis HAS were compared with the recombinant proteins expressed in Escherichia coli membranes. Based on their amino acid sequences, the masses of these four proteins as monomers are approximately 48 kDa. In all cases, loss of enzyme activity was a simple single exponential function with increasing radiation dose. The functional sizes calculated from these data were identical for the four HASs at approximately 64 kDa. In contrast, the sizes of the proteins estimated by the loss of antibody reactivity on Western blots were essentially identical at 41 kDa for the four HAS species, consistently lower than the functional size by approximately 23 kDa. Matrix-assisted laser desorption time of flight mass spectrometry analysis of purified S. pyogenes HAS-H6 and S. equisimilis HAS-H6 gave masses that differed by <0.07% from the predicted monomer sizes, which confirms that neither protein is posttranslationally modified or covalently attached to another protein. Ongoing studies indicate that the purified HAS enzymes require cardiolipin (CL) for maximal activity and stability. When irradiated membranes were detergent solubilized and the extracts were incubated with exogenous CL, the residual level of HAS activity increased. Consequently, the calculated functional size decreased by approximately 23 kDa to the expected size of the HAS monomer. The approximately 23-kDa larger size of the functional HAS enzyme, compared with the HAS monomer, is due, therefore, to CL molecules. We propose that the active streptococcal HA synthases are monomers in complex with approximately 16 CL molecules.

IgA induced activation of human mesangial cells: independent of FcalphaR1 (CD 89).

BACKGROUND: IgA nephropathy (IgAN) is characterized by deposition of polymers of IgA1 in the mesangium, accumulation of mesangial matrix and mesangial cell proliferation. Activation of the mesangial cell by IgA, via an IgA receptor, may be an initiating event in the pathology of IgAN. METHODS: We examined the ability of radiolabeled, normal serum IgA1 to bind human mesangial cells (HMC). Activation of HMC by monomeric (mIgA1) and heat aggregated IgA1 (AIgA1) was compared by Northern analysis of c-jun expression. The expression of FcalphaR1 (CD89) mRNA on our cultured mesangial cells was also assessed by Northern analysis, reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry. RESULTS: 125I-mIgA1 and 125I-AIgA1 bound to HMC in a dose-dependent, saturable manner with similar affinities. There were 1.2 x 10(6) binding sites per cell, with an affinity constant of 2.3 x 10(6) M(-1). AIgA1 induced c-jun expression in a time and dose-dependent manner (2.4-fold above baseline after 60 min exposure to AIgA1 200 microg/ml) while mIgA1 had no effect on c-jun expression. No message for CD 89 was detectable in quiescent or AIgA1 stimulated HMC by Northern analysis or RT-PCR using several primer sequences based on the sequence of U937 FcalphaR cDNA. Flow cytometry on the mesangial cells, using My 43, a monoclonal antibody to FcalphaR1 confirmed that CD 89 was not present on the cell. CONCLUSION: These results demonstrate that HMC bind mIgA1 and AIgA1 with similar affinity. However, activation of HMC requires an aggregated form of IgA1. These processes are independent of FcalphaR1, suggesting the presence of a new IgA receptor on mesangial cells.

A specific antibody to the carbohydrate recognition domain of the asialoglycoprotein receptor RHL1 subunit does not react with RHL2/3 but blocks ligand binding.

The rat asialoglycoprotein receptor (ASGPR) is believed to be a hetero-oligomer composed of three subunits, designated rat hepatic lectin 1, 2, and 3 (RHL1, 2, and 3). The carbohydrate recognition domains (CRDs) of RHL1 and RHL2/3 are 56% identical. We developed a polyclonal antibody that specifically recognizes the CRD of RHL1 but not RHL2/3. When purified ASGPRs were bound to ligand-Sepharose, the CRD of RHL1, but not RHL2 or RHL3, was resistant to digestion with subtilisin. Antibody against purified RHL1 CRD recognized only RHL1 in Western blot analysis of crude cell extracts or purified receptors without detectable cross-reaction to RHL2/3. Although it does not recognize the CRD of RHL2 or RHL3, this antibody specifically inhibited 80-90% of the cell surface or total cellular 125I-ASOR binding to isolated rat hepatocytes and > 90% of ligand binding to purified rat ASGPRs. The antibody also immunoprecipitates active ASGPRs containing all three RHL subunits. The results indicate that homo-oligomeric RHL2/3 complexes, able to bind ASOR, do not form on hepatocytes by subunit rearrangement.

Ethanol feeding causes inactivation of both state 1 and state 2 rat hepatic asialoglycoprotein receptors.

Previous studies have shown that ethanol feeding in rats causes inactivation and redistribution of approximately 50% of the total asialoglycoprotein receptors (ASGPRs) in hepatocytes (Tworek et al., J. Biol. Chem. 271:2531, 1996), and that two equal populations of hepatic ASGPRs mediate ligand uptake and processing via two functionally different pathways (Weigel in Glycoconjugates: Composition, Structure and Function, Marcel Dekker, 1992, p. 421). The purpose of this study was to determine if ethanol feeding causes preferential inactivation of only one of these two ASGPR populations, which have been designated state 1 and state 2 ASGPRs. The state 2, but not state 1, ASGPRs are inactivated in isolated hepatocytes by a variety of drugs and inhibitors. State 2 ASGPRs can also be inactivated in permeable cells by ATP treatment and then reactivated by treatment with fatty acyl coenzyme As. In the present study, permeable cell assays for state 2 ASGPR inactivation and reactivation were used to assess whether hepatocytes from ethanol-fed rats contain inactive state 2 ASGPRs. The results show that preferential inactivation of one ASGPR population does not occur after ethanol feeding. That inactive ASGPRs could not be reactivated by treatment with palmitoyl-coenzyme A to a greater extent in ethanol-fed versus control cells indicates there is not a larger pool of inactivated state 2 ASGPRs in treated cells. We conclude that ethanol feeding causes equal inactivation of both state 1 and state 2 ASGPRs. Ethanol feeding may represent the first treatment found to inactivate state 1 ASGPRs.

Production of neurotoxic factors by brain endothelium in Alzheimer's disease.

The cerebral vasculature is central to the maintenance of the neuronal microenvironment. We have previously demonstrated that brain microvessels in Alzheimer's disease produce high, potentially toxic, levels of nitric oxide. It is our hypothesis that neuronal injury in Alzheimer's disease occurs because an abnormal endothelium secretes factors that are toxic to neurons. In this study, we report that inhibition of protein kinase C in endothelial cells causes release of a factor that is toxic to neurons in vitro. Our results demonstrate that this endothelium-derived toxic factor is soluble, heat-labile, susceptible to proteolysis, and loses activity with repeated freeze-thawing. The molecular weight of this putative protein is between 10 and 50 kDa, and 8 hours are required after protein kinase C inhibition to detect the endothelium-derived toxic factor in the media. Finally, the endothelium-derived toxic factor kills neurons within 2 hours, suggesting that cell death occurs via necrosis, not apoptosis. These data support the notion that endothelial cells can create an injurious microenvironment for neurons by producing molecules with noxious properties. Altered/dysfunctional endothelial cells in the cerebral microcirculation could be a novel, unexplored source of neurotoxic factors in Alzheimer's disease.

Identification of a 175 kDa protein as the ligand-binding subunit of the rat liver sinusoidal endothelial cell hyaluronan receptor.

The rat liver sinusoidal endothelial cell (LEC) hyaluronan (HA) receptor was previously identified using a photoaffinity HA derivative (J. Biol. Chem., 267, 20451-20456, 1992). Two polypeptides with M(r) = 175,000 and 166,000, were consistently crosslinked, suggesting that the LEC HA receptor is an oligomer. Whether one or both subunits participate in HA binding, was not determined. Here we investigate the HA-subunit interactions and the potential oligomeric nature of the LEC HA receptor. When Sephacryl-400 gel filtration chromatography was used to enrich the HA receptor, the 175 kDa polypeptide was the major band seen by SDS-PAGE analysis. Little staining was seen at 166 kDa, suggesting that the 175 kDa protein could be separated from the 166 kDa protein and still retain HA-binding activity. A ligand blot assay was used to determine if each individual subunit could bind HA. LEC proteins were separated by nonreducing SDS-PAGE, and then immobilized onto nitrocellulose. 125I-HA bound to a 175 kDa polypeptide but not to the 166 kDa protein. A high molecular weight band of approximately 300,000 also bound 125I-HA. 125I-HA binding to the 175 and 300 kDa proteins showed the same specificity of competition with a panel of carbohydrates as the bona fide LEC HA receptor. The 175 kDa HA-binding subunit may be nonglobular (asymmetric), since its apparent size by SDS-PAGE is dependent on the polyacrylamide gel pore size; M(r) increases as porosity decreases. LECs were crosslinked to an 125I-labeled photoaffinity HA derivative and the HA saccharides were then released with hyaluronidase. After SDS-PAGE without reduction, radiolabeled bands were seen at 175 and 166 kDa (3:1 ratio), and a high MW (approximately 300,000) species was also detected. These data support an oligomeric model of the LEC HA receptor, and show that the 175 kDa protein possesses HA-binding activity independent from the 166 kDa polypeptide.