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.
active-site cysteines; elongation rate; polymer length; size control; size control model
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).
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.
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.
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.
coated pit mediated; conformation; glycosaminoglycan turnover; HA binding affinity; Stabilin-2
Significant increases in hyaluronan (HA) are readily detectable in chick choroids during the process of recovery from form-deprivation myopia. This HA accumulation is initiated by a rapid increase in choroidal expression of the HAS2 gene in response to myopic defocus.
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.
The hyaluronic acid receptor for endocytosis (HARE)/ Stabilin-2 is the primary systemic scavenger receptor for hyaluronan (HA), the chondroitin sulfates (CS), dermatan sulfate (DS), and nonglycosaminoglycan (GAG) ligands such as acetylated low-density lipoprotein (AcLDL), pro-collagen propeptides, and advanced glycation end products. We recently discovered that HARE is also a systemic scavenger receptor for heparin (Hep) (Harris EN, Weigel JA, Weigel PH. 2008. The human hyaluronan receptor for endocytosis [HARE/Stabilin-2] is a systemic clearance receptor for heparin. J Biol Chem. 283:17341–17350). Our goal was to map the binding sites of eight different ligands within HARE. We used biotinylated GAGs and radio-iodinated streptavidin or AcLDL to assess the binding activities of ligands directly or indirectly (by competition with unlabeled ligands) in endocytosis assays using stable cell lines expressing the 315 or 190 kDa HA receptor for endocytosis (315- or 190-HARE) isoforms, and ELISA-like assays, with purified recombinant soluble 190-HARE ecto-domain. For example, Hep binding to HARE was competed by DS, CS-E, AcLDL, and dextran sulfate, but not by other CS types, HA, dextran, or heparosan. 125I-AcLDL binding to HARE was partially competed by Hep and dextran sulfate, but not competed by HA. Two ligands, DS and CS-E, competed with both Hep and HA to some degree. Hep and HA binding or endocytosis is mutually inclusive; binding of these two GAGs occurs with functionally separate, noncompetitive, and apparently noninteracting domains. Thus, HARE binds to HA and Hep simultaneously. Although the domain(s) responsible for Hep binding remains unknown, the Link domain was required for HARE binding to HA, CS-A, CS-C, and CS-D. These results enable us to outline, for the first time, a binding activity map for multiple ligands of HARE.
chondroitin sulfate; endocytosis; glycosaminoglycan turnover; heparin; Stabilin-2
Extracellular matrix changes occur in many heart valve pathologies. For example, myxomatous mitral valves are reported to contain excess proteoglycans (PGs) and hyaluronan (HA). However, it is unknown which specific PGs are altered in myxomatous valves. Because PGs perform varied functions in connective tissues, this study was designed to identify and localize three matrix-associated PGs as well as HA and the HA receptor for endocytosis (HARE) within myxomatous and normal mitral valves.
Human mitral posterior leaflets (control n=6−9, myxomatous n=14−21, mean age 61 for all groups) were histochemically stained for PG core proteins, HA, and HARE. Stain intensity was semi-quantitatively graded to determine differences in marker abundance betweennormal and myxomatous valves. The PGs were localized to different regions of the leaflet by correspondence to parallel Movat stained sections
The PGs decorin, biglycan and versican were more abundant in myxomatous valves than in normal controls (p<0.03). There was a gender effect on PG presence but no age related trends were observed. HA and HARE were distributed throughout all valves. There was no significant difference in HA between groups, but HARE expression was reduced in myxomatous valves compared to normal controls (p<0.002).
Excess decorin, biglycan and versican may be associated with the remodeling of other matrix components in myxomatous mitral valves. Decreased expression of HARE in myxomatous valves suggests that HA metabolism could be altered in myxomatous mitral valve disease. These finding contribute towards elucidating the pathogenesis of myxomatous mitral valve disease and developing potential new therapies.
Myxomatous mitral valve; Proteoglycan; Hyaluronan; Remodeling; Extracellular matrix
The Hyaluronic Acid Receptor for Endocytosis (HARE)/Stabilin-2 is the primary systemic scavenger receptor for hyaluronan (HA), the chondroitin sulfates (CS), dermatan sulfate (DS), and non-glycosaminoglycan (GAG) ligands such as acetylated low density lipoprotein (AcLDL), pro-collagen propeptides, and advanced glycation end-products. We recently discovered that HARE is also a systemic scavenger receptor for heparin (Harris et al, J. Biol. Chem. 283, 2008;ePub Apr 22). Our goal was to map the binding sites of eight different ligands within HARE. We used biotinylated GAGs and radio-iodinated streptavidin or AcLDL to assess the binding activities of ligands directly or indirectly (by competition with unlabeled ligands) in endocytosis assays using stable cell lines expressing the 315- or 190-HARE isoforms, and ELISA-like assays, with purified recombinant soluble 190-HARE ecto-domain. For example, Hep binding to HARE was competed by DS, CS-E, AcLDL, and dextran sulfate, but not by other CS types, HA, dextran, or heparosan. 125I-AcLDL binding to HARE was partially competed by Hep and dextran sulfate, but not competed by HA. Two ligands, DS and CS-E, competed with both Hep and HA to some degree. Heparin and HA-binding or endocytosis are mutually inclusive; binding of these two GAGs occurs with functionally separate, non-competitive, and apparently non-interacting domains. Thus, HARE binds to HA and Hep simultaneously. Although, the domain(s) responsible for Hep-binding remains unknown, the Link domain was required for HARE binding to HA, CS-A, CS-C, and CS-D. These results enable us to outline, for the first time, a binding-activity map for multiple ligands of HARE.
chondroitin sulfate; endocytosis; glycosaminoglycan turnover; heparin; Stabilin-2
reducing end; nonreducing end; direction of synthesis; polysaccharide; Glycosyltransferase; glycosaminoglycan
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 pKa values at pH 6.6 and 11.8. As the pH was increased from ~6.5, both Km and Vmax increased until pH ~10.5, above which the kinetic constants gradually declined. Nonetheless, the overall catalytic constant (120/sec) was essentially unchanged from pH 6.5 to pH 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 Ea 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 Vmax at 21, 6.5, and 3.5 mM, respectively. The apparent Ki values for ethylene glycol, glycerol, and sucrose were, respectively, 4.5, 3.3 and 1.2 mM.
streptococcal; kinetics; pH; viscogens; temperature; divalent cations; CLm cardiolipin; ECM, extracellular matrix; GAG, glycosaminoglycan; HA, hyaluronic acid, hyaluronate, hyaluronan; HAS, HA synthase; seHAS, Streptococcus equisimilis HAS; PBS, phosphate buffered saline; Tris, trishydroxymethylamino methane; TBS, tris-buffered saline; TBST, tris-buffered saline containing 0.05% Tween20
SEC-MALLS analyses of E. coli membranes expressing Streptococcus equisimilis hyaluronan synthase (seHAS) demonstrated an inherent artifact (10–100 MDa) that co-eluted with HA, and skewed the apparent weight-average mass of HA to erroneously high values. Briefly heating samples to 65–75°C eliminated this artifact and increased the yield of recovered HA, due to the release of HA chains that were attached to membrane-bound HAS. Inclusion of alkaline phosphatase, which removed UDP produced during the reaction, improved the linearity of HA synthesis - even at high substrate utilization. Surprisingly, addition of EDTA, to chelate Mg+2 ions, did not completely stop the HAS reaction at 30°C or at 4°C. The best conditions for stopping the reaction without altering SEC-MALLS profiles of the product HA were to chill samples on ice in the presence of both EDTA and UDP. Even with excess substrate, the maximum size of product HA decreased as the enzyme concentration increased. Therefore, the maximum HA size made by HAS was determined by extrapolation to zero enzyme concentration. Using the above conditions, membrane-bound seHAS synthesized a cohort of HA products that steadily increased in weight-average molar mass, reaching a final maximal steady-state size of 4–6 MDa within 2–4 hours.
streptococcal; hyaluronan synthase; light scattering; size distribution; membranes; molar mass
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 wildtype enzyme (Kumari et al., J. Biol. Chem. 277, 13943, 2002). 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 that Cys367 is not. Substrate protection studies of wildtype 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 crosslink vicinyl sulfhydryl groups, also supported the conclusion that Cys262 and Cys281 are close enough to be crosslinked. 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.
Sulfhydryl reagents; N-ethylmaleimide; enzyme inhibition; Cysteine modification; site directed mutagenesis; DTE, dithioerythritol; HA, hyaluronan or hyaluronic acid; HAS, HA synthase; NEM, N-ethylmaleimide; PBS, phosphate buffered saline; seHAS, Streptococcus equisimilis HAS; spHAS, Streptococcus pyogenes HAS
The hasA gene from Streptococcus equisimilis, which encodes the enzyme hyaluronan synthase, has been expressed in Bacillus subtilis, resulting in the production of hyaluronic acid (HA) in the 1-MDa range. Artificial operons were assembled and tested, all of which contain the hasA gene along with one or more genes encoding enzymes involved in the synthesis of the UDP-precursor sugars that are required for HA synthesis. It was determined that the production of UDP-glucuronic acid is limiting in B. subtilis and that overexpressing the hasA gene along with the endogenous tuaD gene is sufficient for high-level production of HA. In addition, the B. subtilis-derived material was shown to be secreted and of high quality, comparable to commercially available sources of HA.
We recently purified the rat liver hyaluronan receptor for endocytosis (HARE) and found abundant expression of 175- and ∼300-kDa HARE species in sinusoidal endothelial cells of the liver, spleen, and lymph nodes. We report herein the first cloning and functional expression of the rat 175-kDa HARE. Peptide sequences were obtained from the purified 175-kDa HARE, and degenerate oligonucleotide primers were designed for reverse transcription-polymerase chain reaction and cDNA cloning. Results of 5′-rapid amplification of cDNA ends, Northern analysis, N-terminal sequence, and antibody reactivity analyses indicated the absence of mRNA directly encoding the 175-kDa HARE. This protein is most likely derived from a larger precursor. Accordingly, we constructed an artificial 4.7-kb cDNA encoding the 1431 amino acid 175-kDa HARE. The predicted type I membrane protein has a mass of 156,393 Da and a pI of 7.86. The 175-kDa HARE cDNA, fused to the N-terminal leader sequence of the Ig κ-chain, was transfected transiently into COS-7 cells and stably into SK-Hep-1 cells, respectively, to assess hyaluronan or hyaluronic acid (HA)-binding activity and endocytosis. In both cases, HARE expression and HA-binding activity were detected. Furthermore, stable SK-175HARE cells demonstrated specific endocytosis of 125I-HA and receptor recycling. Fluorescence-activated cell sorting analysis confirmed that recombinant HARE was expressed on the cell surface and that fluorescent HA uptake was inhibited by a specific blocking monoclonal antibody against HARE. Additionally, HARE was substantially colocalized with clathrin, but not with internalized HA that was delivered to lysosomes. The results confirm that recombinant 175-kDa HARE is an authentic endocytic receptor for HA and that this receptor can function independently of the ∼300-kDa HARE. HARE is the first functionally identified member of a protein family that shares a similar organization of Fasciclin, epidermal growth factor-like, Xlink, and transmembrane domains.