Several studies have convincingly shown that in chicks, compensation for imposed focus involves immediate changes in choroid thickness. The molecular events associated with choroidal thickening and ...the regulation of the choroidal response are largely unknown.
Form-deprivation myopia was induced in the right eyes of 2-day-old chicks by the application of translucent occluders for 10 days and was followed by unrestricted vision for an additional 1 to 20 days (recovery). Individual choroids were isolated from treated and control eyes and used for reverse transcription-quantitative PCR, hyaluronan (HA) localization with biotinylated hyaluronic acid binding protein (b-HABP), and analyses of HA size and concentration by size exclusion chromatography-multiangle laser light scattering (SEC-MALLS).
HAS2 gene expression increased significantly after 6 hours of unrestricted vision (>7-fold) and peaked at 24 hours (>9-fold). In untreated eyes, HA was localized to perivascular sheaths of larger choroidal blood vessels; however, after 4 to 15 days of recovery, intense labeling for HA was detected throughout the thickened choroidal stroma. Analyses of choroidal HA by SEC-MALLS indicated that HA concentration was significantly increased in recovering choroids compared with controls after 4 to 8 days of recovery (≈3.5-fold).
Newly synthesized HA accumulates in the choroidal stroma of recovering eyes and is most likely responsible for the stromal swelling observed during recovery from myopia. This HA accumulation is initiated by a rapid increase in choroidal expression of the HAS2 gene in response to myopic defocus.
Previous studies reached different conclusions about whether class I hyaluronan synthases (HASs) elongate hyaluronic acid (HA) by addition to the reducing or the nonreducing end. Here we used two ...strategies to determine the direction of HA synthesis by purified class I HASs from Streptococcus equisimilis and Streptococcus pyogenes. In the first strategy we used each of the two UDP-sugar substrates separately to pulse label either the beginning or the end of HA chains. We then quantified the relative rates of radioactive HA degradation by treatment with β-glycosidases that act at the nonreducing end. The results with both purified HASs demonstrated that HA elongation occurred at the reducing end. In the second strategy, we used purified S. equisimilis HAS, UDP-glucuronic acid, and UDPβ-32P-Glc-NAc to radiolabel nascent HA chains. Under conditions of limiting substrate, the 32P-labeled products were separated from the substrates by paper chromatography and identified as HA-32PUDP saccharides based on their degradation by snake venom phosphodiesterase or hyaluronidase and by their binding to a specific HA-binding protein. The 32P radioactivity was chased (released) by incubation with unlabeled UDP-sugars, showing that the HA-UDP linkages turn over during HA biosynthesis. In contrast, HA-32PUDP products made by the purified class II Pasteurella multocida HAS were not released by adding unlabeled UDP-sugars, consistent with growth at the nonreducing end for this enzyme. The results demonstrate that the streptococcal class I HAS enzymes polymerize HA chains at the reducing end.
We identified two conserved polar amino acids within different membrane domains (MD) of Streptococcus equisimilis hyaluronan synthase (seHAS), Lys48 in MD2 and Glu327 in MD4. In eukaryotic HASs, the ...position of the Glu is very similar and the Lys is replaced by a conserved polar Gln. To assess whether Lys48 and Glu327 interact or influence seHAS activity, we investigated the effects of changing Lys48 to Arg or Glu and Glu327 to Lys, Asp, or Gln. Mutants, including a double switch variant with Lys48 and Glu327 exchanged, were expressed and assayed in Escherichia coli membranes. SeHAS(E327Q) and seHAS(E327K) were expressed at low levels, whereas seHAS(E327D) and the Lys48 mutants were expressed well. The specific enzyme activities (relative to wild type) were 17 and 7% for the K48R and K48E mutants and 26 and 38% for the E327Q and E327D mutants, respectively. In contrast, seHAS(E327K) showed only 0.16% of wild-type activity but was rescued over 46-fold by changing Lys48 to Glu. Expression of the seHAS(E327K,K48E) protein was also rescued to near wild-type levels. Based on size exclusion chromatography coupled to multiangle laser light scattering analysis, all the variants synthesized hyaluronan (HA) of smaller weight-average molar mass than wild-type enzyme (3.6 MDa); the smallest HA (∼0.6 MDa) was made by seHAS(E327K,K48E) and seHAS(K48E). The results indicate that Glu327 within MD4 is a critical residue for the stability of seHAS, that it may interact with Lys48 within MD2, and that these residues are involved in the ability of HAS to synthesize very large HA.
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.
Hyaluronan synthase (HAS) utilizes UDP-GlcUA and UDP-GlcNAc in the presence of Mg2+ to form the GAG hyaluronan (HA). The purified HAS from Streptococcus equisimilis (seHAS) shows high fidelity in ...that it only polymerizes the native substrates, UDP-GlcNAc and UDP-GlcUA. However, other uridinyl nucleotides and UDP-sugars inhibited enzyme activity, including UDP-GalNAc, UDP-Glc, UDP-Gal, UDP-GalUA, UMP, UDP, and UTP. Purified seHAS was ∼40% more active in 25 mM, compared to 50 mM, PO4 in the presence of either 50 mM NaCl or KCl, and displayed a slight preference for KCl over NaCl. The pH profile was surprisingly broad, with an effective range of pH 6.5−11.5 and the optimum between pH 9 and 10. SeHAS displayed two apparent pK a values at pH 6.6 and 11.8. As the pH was increased from ∼6.5, both K m and V max increased until pH ∼ 10.5, above which the kinetic constants gradually declined. Nonetheless, the overall catalytic constant (120/s) was essentially unchanged from pH 6.5 to 10.5. The enzyme is temperature labile, but more stable in the presence of substrate and cardiolipin. Purified seHAS requires exogenous cardiolipin for activity and is very sensitive to the fatty acyl composition of the phospholipid. The enzyme was inactive or highly activated by synthetic cardiolipins containing, respectively, C14:0 or C18:1(Δ9) fatty acids. The apparent E act for HA synthesis is 40 kJ (9.5 kcal/mol) disaccharide. Increasing the viscosity by increasing concentrations of PEG, ethylene glycol, glycerol, or sucrose inhibited seHAS activity. For PEGs, the extent of inhibition was proportional to their molecular mass. PEGs with average masses of 2.7, 11.7, and 20 kg/mol caused 50% inhibition of V max at 21, 6.5, and 3.5 mM, respectively. The apparent K i values for ethylene glycol, glycerol, and sucrose were, respectively, 4.5, 3.3, and 1.2 mM.
Previous radiation inactivation and enzyme characterization studies demonstrated that the Streptococcus equisimilis hyaluronan synthase (seHAS) is phospholipid-dependent and that cardiolipin (CL) is ...the best phospholipid for enzyme activation. Here we investigated the ability of seHAS, purified in the absence of added lipid, to be activated by synthetic phosphatidic acid (PA), phosphatidylserine, or CL lipids containing fatty acyl chains of different length or different numbers of double bonds. The most effective lipid was tetraoleoyl CL (TO-CL), whereas tetramyristoyl CL (TM-CL) was ineffective. None of the phosphatidylserine species tested gave significant activation. PAs containing C10 to C18 saturated acyl chains were not effective activators, and neither were oleoyl lyso PA, dilinoleoyl PA, or PA containing one oleoyl chain and either a palmitoyl or stearoyl chain. In contrast, dioleoyl PA stimulated seHAS ∼10-fold, to ∼20% of the activity observed with TO-CL. The tested acidic lipids such as PA and CL activated the enzyme most efficiently if they contained only oleic acid. Mixing experiments showed that the enzyme interacts preferentially with TO-CL in the presence of TM-CL. Similarly, seHAS incorporated into phosphotidylcholine-based liposomes showed increasing activity with increasing TO-CL, but not TM-CL, content. Inactivation of membrane-bound seHAS by solubilization with Nonidet P-40 was prevented by TO-CL, but not TM-CL. The pH dependence of seHAS in the presence of synthetic or naturally occurring CLs showed the same pattern of lipid preference between pH 6 and 10.5. Unexpectedly, HAS showed lipid-independent activity at pH 11.5. The results suggest that Class I HAS enzymes are lipid-dependent and that assembly of active seHAS-lipid complexes has high specificity for the phospholipid head group and the nature of the fatty acyl chains.
The clearance of hyaluronan (HA) and chondroitin sulfates from the circulating blood and lymph in the body is mediated by the membrane-bound HA receptor for endocytosis (HARE). Previously, we found ...that two HARE species of ∼175 kDa and ∼300 kDa are abundant in the sinusoidal endothelial cells in rat liver, spleen, and lymph nodes (Zhou et al. 2000, J. Biol. Chem., 275, 37733–37741). In the present study, immunocytochemical analysis of human tissues showed a similar pattern with abundant expression of HARE in the sinusoidal endothelial cells of human liver, spleen, and lymph nodes. The two human HARE proteins were immunoaffinity-purified from human spleen. Each protein was recognized in western blots using several anti-rat HARE monoclonal antibodies and was able to bind 125I-HA specifically. In nonreducing SDS–PAGE, these two human HARE species migrated at ∼190 kDa and ∼315 kDa; both proteins are ∼15 kDa larger than the corresponding rat HAREs, although the de-N-glycosylated core proteins are essentially the same mass. After reduction, the human 190-kDa HARE gave a single 196-kDa species, which was not seen in the ∼315-kDa HARE after reduction. The reduced ∼315-kDa HARE yielded two major proteins at ∼250 kDa and ∼220 kDa. We determined the sequence of the human 190-kDa HARE cDNA based on analysis of internal tryptic peptides, as well as RT-PCR and 5′ RACE analyses using human spleen and lymph node cDNA libraries. The human gene that encodes HARE is on chromosome 12.
The hyaluronic acid receptor for endocytosis (HARE)/Stabilin-2 is the primary systemic scavenger receptor for 13 ligands including hyaluronan (HA), heparin and chondroitin sulfates. Most ...ligand-binding sites are within the 190 kDa isoform, which contains ∼25 kDa of N-glycans and is the C-terminal half of the full-length 315 kDa HARE. Glycoproteomic analyses of purified recombinant human 190-HARE ecto-domain identified a diverse population of glycans at 10 of 17 consensus sites. The most diversity (and the only sialylated structures) occurred at N2280, within the HA-binding Link domain. To determine if these N-glycans are required for HA binding, we created human Flp-In 293 cell lines expressing membrane-bound or soluble ecto-domain variants of 190-HARE(N2280A). Membrane-bound HARE lacking Link domain N-glycans mediated rapid HA endocytosis, but purified 190-HARE(N2280A) ecto-domain showed little or no HA binding in ELISA-like, HA-HARE pull-down assays or by surface plasmon resonance analysis (which detected very high apparent affinity for 190-HARE ecto-domain binding to HA; Kd = 5.2 nM). The results indicate that Link domain N-glycans stabilize interactions that facilitate HA binding to HARE.
The hepatic asialoglycoprotein receptor (ASGP-R) internalizes desialylated glycoproteins via the clathrin-coated pit pathway
and mediates their delivery to lysosomes for degradation. The human ASGP-R ...contains two subunits, H1 and H2. Cytoplasmic residues
Cys 36 in H1, as well as Cys 54 and Cys 58 in H2 are palmitoylated (Zeng, F.-Y., and Weigel, P.âH. (1996) J. Biol. Chem . 271, 32454). In order to study the function(s) of ASGP-R palmitoylation, we mutated these Cys residues to Ser and generated
stably transfected SK-Hep-1 cell lines expressing either wild-type or nonpalmitoylated ASGP-Rs. Compared with wild-type ASGP-Rs,
palmitoylation-defective ASGP-Rs showed normal ligand binding, intracellular distribution and trafficking patterns, and pH-induced
dissociation profiles in vitro . However, continuous ASOR uptake, and the uptake of prebound cell surface ASOR were slower in cells expressing palmitoylation-defective
ASGP-Rs than in cells expressing wild-type ASGP-Rs. Unlike native ASGP-Rs in hepatocytes or hepatoma cells, which mediate
endocytosis via the clathrin-coated pit pathway and are almost completely inhibited by hypertonic medium, only â¼40% of the
ASOR uptake in SK-Hep-1 cells expressing wild-type ASGP-Rs was inhibited by hyperosmolarity. This result suggests the existence
of an alternate nonclathrin-mediated internalization pathway, such as transcytosis, for the entry of ASGP-R·ASOR complexes
into these cells. In contrast, ASOR uptake mediated by cells expressing palmitoylation-defective ASGP-Rs showed only a marginal
difference under hypertonic conditions, indicating that most of the nonpalmitoylated ASGP-Rs were not internalized and processed
normally through the clathrin-coated pit pathway. Furthermore, cells expressing wild-type ASGP-Rs were able to degrade the
internalized ASOR, whereas ASOR dissociation was impaired and degradation was barely detectable in cells expressing nonpalmitoylated
ASGP-Rs. We conclude that palmitoylation of the ASGP-R is required for its efficient endocytosis of ligand by the clathrin-dependent
endocytic pathway and, in particular, for the proper dissociation and delivery of ligand to lysosomes.
We describe a sensitive assay for detection of active hyaluronan synthases (HASs) capable of synthesizing hyaluronan (HA) without use of radioactive uridine 5′-diphosphate sugar precursors. The HAS ...capture assay is based on the binding of a biotinylated HA binding protein (bHABP) to HA chains that are associated with HAS and the subsequent capture of bHABP–HA–HAS complexes with streptavidin–agarose. Specific HAS proteins (e.g., HAS1, not HAS2 or HAS3) captured in this pull-down approach are readily immunodetected by Western blot analysis using appropriate antibodies. The assay was used to detect active HAS proteins in cell membranes, purified recombinant Streptococcus equisimilis HAS (SeHAS), and in vitro translated human HAS1 or SeHAS. The HAS capture assay was also used to assess the fraction of HAS molecules that were active, which cannot be done using standard assays for synthase activity. Assay sensitivity for detection of purified SeHAS is <1 pmol.
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