Examination of corneal proteoglycans and glycosaminoglycans by rotary shadowing and electron microscopy

Proteoglycans (PGs) from cornea and their relevant glycosaminoglycan (GAG) chains, dermatan sulphate (DS) and keratin sulphate (KS), were examined by electron microscopy following rotary shadowing, and compared with hyaluronan (HA), chondroitin sulphate (CS), alginate, heparin, heparan sulphate (HS) and methyl cellulose. Corneal DS PG had the tadpole shape previously seen in scleral DS FG, and the images from corneal KS PG could be interpreted similarly, although the GAG (KS) chains were very much fainter than those of DS PG GAG. Isolated GAG (KS, DS, CS, HA, etc.) examined in the same way showed images that decreased very significantly in clarity and contrast, in the sequence HA greater than DS greater than CS greater than KS. The presence of secondary and tertiary structures in the GAGs may be at least partly responsible for these variations. HA appeared to be double stranded, and DS frequently self-aggregated, KS and HS showed tendencies to coil into globular shapes. It is concluded that it is unsafe to assume the absence of GAGs, based on these techniques, and quantitative measurements of length may be subject to error. The results on corneal DS PG confirm and extend the hypothesis that PGs specifically associated with collagen fibrils are tadpole shaped.     

Glycosaminoglycans of Rat Cerebellum: II. A Developmental Study

Total and individual glycosaminoglycans (GAGs) were determined in rat cerebellum in tissue explants at various postnatal ages. The major constituents of GAGs were chondroitin sulfate (CS), hyaluronic acid (HA), and heparan sulfate (HS). Dermatan sulfate (DS) and keratan sulfate (KS) could not be detected and therefore each amounts to less than 5% of all GAGs at all ages studied. HA was the prominent GAG during postnatal development and only a minor constituent at adult ages, whereas CS was the predominant GAG in adulthood. HS remained relatively constant throughout development. The incorporation of [3H]glucosamine into individual GAGs was highest for HS at postnatal day 6, whereas HA showed intermediate and CS the lowest levels of incorporation during the first postnatal week. All major GAGs showed the lowest incorporation values at adult ages.     

Characterization of glycosaminoglycans from human normal and scoliotic nasal cartilage with particular reference to dermatan sulfate.

The composition and the distribution of glycosaminoglycans (GAGs) present in normal human nasal cartilage (HNNC), were examined and compared with those in human scoliotic nasal cartilage (HSNC). In both tissues, hyaluronan (HA), keratan sulfate (KS) and the galactosaminoglycans (GalAGs)--chondroitin sulfate (CS) and dermatan sulfate (DS)--were identified. The overall GAG content in HSNC was approx. 30% higher than the HNNC. Particularly, a 114% increase in HA, and 46% and 86% in KS and DS, respectively, was recorded. CS was the main type of GAG in both tissues with no significant compositional difference. GalAG chains in HSNC exhibited an altered disaccharide composition which was associated with significant increases of non-sulfated and 6-sulfated disaccharides. DS, which was identified and quantitated for the first time in HNNC and HSNC, contained low amounts of iduronic acid (IdoA), 18% and 28% respectively. In contrast to other tissues, where IdoA residues are organized in long IdoA rich repeats, the IdoA residues of DS in human nasal cartilage seemed to be randomly distributed along the chain. DS chains in HSNC were of larger average molecular size than those from HNNC. These results clearly indicate the GAG content and pattern in both HNNC and HSNC and demonstrate that scoliosis of nasal septum cartilage is related to quantitative and structural modifications at the GAG level.     

Effect of sulfated GAGs on the expression and activation of MMP-2 and MMP-9 in corneal and dermal explant cultures

It has been shown previously that hyaluronan (HA) added to fibroblast and keratocyte cell cultures or corneal explant cultures produces an up-regulation of MMP-2 and MMP-9 expression and activation. Here, we examine the effect of sulfated GAG-s, chondroitin 4 and 6 sulfate (CS4, CS6), dermatan sulfate (DS), keratan sulfate (KS) and heparan sulfate (HS) on MMP-2 and 9 expression and activation under the same culture conditions. It appears that CS4 has only minor effects, KS inhibits MMP-2 activation and CS6, DS and HS increase MMP-2 activation in corneal explant cultures. For skin explant cultures, DS, KS and HS strongly increase MMP-9 activation, whereas KS inhibits and DS increases MMP-2 activation. All these effects can be strongly inhibited by the addition of an antibody to CD44, except CS6 and DS. Activation by these two GAGs was only slightly affected, supporting the contention that the effects of HA, CS4, KS and HS are mediated by one of the isoforms of this CD44 receptor. The physio-pathological significance of these results is discussed for cornea and skin ageing, because of the divergent evolution with in vitro ageing of the relative proportions of GAGs synthesised by these two cell types.     

The ligand-binding profile of HARE: hyaluronan and chondroitin sulfates A, C, and D bind to overlapping sites distinct from the sites for heparin, acetylated low-density lipoprotein, dermatan sulfate, and CS-E

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. (125)I-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.     

Electrophoretic approaches to the analysis of complex polysaccharides

Complex polysaccharides, glycosaminoglycans (GAGs), are a class of ubiquitous macromolecules exhibiting a wide range of biological functions. They are widely distributed as sidechains of proteoglycans (PGs) in the extracellular matrix and at cellular level. The recent emergence of enhanced analytical tools for their study has triggered a virtual explosion in the field of glycomics. Analytical electrophoretic separation techniques, including agarose-gel, capillary electrophoresis (HPCE) and fluorophore-assisted carbohydrate electrophoresis (FACE), of GAGs and GAG-derived oligosaccharides have been employed for the structural analysis and quantification of hyaluronic acid (HA), chondroitin sulfate (CS), dermatan sulfate (DS), keratan sulfate (KS), heparan sulfate (HS), heparin (Hep) and acidic bacterial polysaccharides. Furthermore, recent developments in the electrophoretic separation and detection of unsaturated disaccharides and oligosaccharides derived from GAGs by enzymatic or chemical degradation have made it possible to examine alterations of GAGs with respect to their amounts and fine structural features in various pathological conditions, thus becoming applicable for diagnosis. In this paper, the electromigration procedures developed to analyze and characterize complex polysaccharides are reviewed. Moreover, a critical evaluation of the biological relevance of the results obtained by these electrophoresis approaches is presented.     

Glycosaminoglycans from bovine eye vitreous humour and interaction with collagen type II

Glycosaminoglycans (GAGs) play an important role in stabilizing the gel state of eye vitreous humour. In this study, the composition of GAGs present in bovine eye vitreous was characterized through disaccharide analysis by liquid chromatography-mass spectrometry. The interaction of GAGs with collagen type II was assessed using surface plasmon resonance (SPR). The percentage of hyaluronic acid (HA), chondroitin sulfate (CS) and heparan sulfate (HS), of total GAG, were 96.2%, 3.5% and 0.3%, respectively. The disaccharide composition of CS consisted of 4S (49%), 0S (38%) 6S (12%), 2S6S (1.5%) and 2S4S (0.3%). The disaccharide composition of HS consisted of 0S (80%), NS2S (7%), NS (7%), 6S (4%), NS6S (2%), and TriS, 2S and 4S6S (each at 0.1%). The average molecular weights of CS and HS were 148 kDa and 204 kDa, respectively. SPR reveals that collagen type II binds to heparin (primarily composed of TriS) with a binding affinity (K _D) of 755 nM and interacts with other GAGs, including CSB and CSE. Both bovine vitreous CS and HS interact with collagen type II, with vitreous HS showing a higher binding affinity.     

Intra- and extracellular localization of hyaluronic acid and proteoglycan constituents (chondroitin sulfate, keratan sulfate, and protein core) in articular cartilage of rabbit tibia.

To demonstrate the intra- and extracellular localization of hyaluronic acid (HA) in articular cartilage of the rabbit tibia, biotinylated HA binding region, which specifically binds to the HA molecule, was applied to the tissue. In comparison with the localization of HA, that of chondroitin sulfate (CS), keratan sulfate (KS), and the protein core (PC) of the proteoglycan was examined by immunohistochemistry. Strong positive staining for HA was detected in chondrocytes located in the transition between the superficial and middle zones of the tissue. Pre-treatment with chondroitinase ABC, keratanase II, or trypsin enhanced the stainability for HA in peri- and intercellular matrices. Immunohistochemistry with or without enzymatic pre-treatment demonstrated that immunoreactivity for CS, KS, and PC was distinctly discerned in chondrocytes and in the extracellular matrix located in the middle and deep zones. In particular, the immunoreactivity for KS and PC was augmented by pre-treatment with chondroitinase ABC not only in chondrocytes but in the extracellular matrix located in the middle and deep zones. Microbiochemical analysis corresponded well with histochemical and immunohistochemical results. These results suggest that HA is abundantly synthesized and secreted in chondrocytes located in the transition between the superficial and middle zones.     

花背蟾蜍角膜早期发育中氨基多糖的电镜细胞化学研究

Glycosaminoglycans (GAGs) and their changes in early corneal development of Bufo raddei Strauch (from stage 16, neural tube, to stage 25, operculum completely closed) were studied with electron microscopic cytochemical method. Results show that synthesis of GAGs changes from non-sulfated to sulfated, and its content increased gradually with the development of cornea. Hyaluronic acid (HA) in each part of cornea begins to increase gradually from stage 16 to 21 (mouth open stage), with its peak at stage 20 (gill circulation stage) to 21, then decreases. In the mean time, contents of dermatam sulfate (DS), chondroitin sulfate (CS), heparan sulfate (HS) and heparin (Hep) increase gradually. It is considered that HA, HS and collagen may be related to the migration of mesenchymal cells, and HA promotes the expansion and hydration of corneal stroma; sulfated GAGs are correlated with dehydration of cornea, cell density and corneal transparency; DS, CS, HS and Hep deposited among collagen fibrils could adjust their arrangement. All these changes would enhance transparency of cornea.     

Modulation of blood coagulation and fibrinolysis by polyamines in the presence of glycosaminoglycans

The effects of polyamines on blood coagulation and fibrinolysis in the presence of glycosaminoglycans (GAGs) were examined because it is known that heparin (HP) interacts with polyamines, especially with spermine. Spermine was able to reverse the prolongation of coagulation time of rabbit plasma caused by HP. The effects of various GAGs on thrombin activity in the presence of anti-thrombin III (AT) were then tested using a synthetic substrate. Inhibition of thrombin activity by GAGs was in the order HP > heparan sulfate (HS) > dermatan sulfate (DS) > chondroitin sulfate (CS) approximately hyaluronan (HA). When these GAGs were fully sulfonated, the inhibitory activity of HS, DS, CS and HA, but not HP, became stronger. The effects of GAGs on thrombin activity were reversed by polyamines, in particular spermine. The EC(50) value of spermine for reversal of HP inhibition was 30-50 microM, and the K(d) value of spermine for heparin was 41.1 microM. Analysis by surface plasmon resonance (SPR) indicated that the interaction between AT and HP was weakened by spermine through its binding to HP. The effect of HP on fibrinolysis was then examined. When Glu-plasminogen and tissue-type plasminogen activator (tPA) were used as enzyme source, HP strongly enhanced the plasmin activity and spermine reversed this effect. Analysis by SPR suggests that the structure of the active site of tPA may be changed through the ternary complex formation of tPA, HP and spermine. The results indicate that blood coagulation was enhanced and fibrinolysis was weakened by spermine in the presence of HP.     

Age-related changes in aggrecan glycosylation affect cleavage by aggrecanase

Aggrecan degradation involves proteolytic cleavage of the core protein within the interglobular domain. Because aggrecan is highly glycosylated with chondroitin sulfate (CS) and keratan sulfate (KS), we investigated whether glycosylation affects digestion by aggrecanase at the Glu(373)-Ala(374) bond. Treatment of bovine aggrecan monomers to remove CS and KS resulted in loss of cleavage at this site, suggesting that glycosaminoglycans (GAGs) play a role in cleavage at the Glu(373)-Ala(374) bond. In contrast, MMP-3 cleavage at the Ser(341)-Phe(342) bond was not affected by glycosidase treatment of aggrecan. Removal of KS, but not CS, prevented cleavage at the Glu(373)-Ala(374) bond. Thus, KS residues may be important for recognition of this cleavage site by aggrecanase. KS glycosylation has been observed at sites adjacent to the Glu(373)-Ala(374) bond in steer aggrecan, but not in calf aggrecan (Barry, F. P., Rosenberg, L. C., Gaw, J. U., Gaw, J. U., Koob, T. J., and Neame, P. J. (1995) J. Biol. Chem. 270, 20516-20524). Interestingly, although we found that aggrecanase degraded both calf and steer cartilage aggrecan, the proportion of fragments generated by cleavage at the Glu(373)-Ala(374) bond was higher in steer than in calf, consistent with our observations using aggrecan treated to remove KS. We conclude that the GAG content of aggrecan influences the specificity of aggrecanase for cleavage at the Glu(373)-Ala(374) bond and suggest that age may be a factor in aggrecanase degradation of cartilage.     

Characterization of the interaction of interleukin-8 with hyaluronan, chondroitin sulfate, dermatan sulfate and their sulfated derivatives by spectroscopy and molecular modeling

The interactions between glycosaminoglycans (GAGs), important components of the extracellular matrix, and proteins such as growth factors and chemokines play critical roles in cellular regulation processes. Therefore, the design of GAG derivatives for the development of innovative materials with bio-like properties in terms of their interaction with regulatory proteins is of great interest for tissue engineering and regenerative medicine. Previous work on the chemokine interleukin-8 (IL-8) has focused on its interaction with heparin and heparan sulfate, which regulate chemokine function. However, the extracellular matrix contains other GAGs, such as hyaluronic acid (HA), dermatan sulfate (DS) and chondroitin sulfate (CS), which have so far not been characterized in terms of their distinct molecular recognition properties towards IL-8 in relation to their length and sulfation patterns. NMR and molecular modeling have been in great part the methods of choice to study the structural and recognition properties of GAGs and their protein complexes. However, separately these methods have challenges to cope with the high degree of similarity and flexibility that GAGs exhibit. In this work, we combine fluorescence spectroscopy, NMR experiments, docking and molecular dynamics simulations to study the configurational and recognition properties of IL-8 towards a series of HA and CS derivatives and DS. We analyze the effects of GAG length and sulfation patterns in binding strength and specificity, and the influence of GAG binding on IL-8 dimer formation. Our results highlight the importance of combining experimental and theoretical approaches to obtain a better understanding of the molecular recognition properties of GAG-protein systems.     

Urinary glycosaminoglycans and glycoproteins in a calcium oxalate crystallization system

This study measures the effects of total urinary glycosaminoglycans (GAGs), glycoproteins (GPs) and individual GAGs on the nucleation rates (Bo), growth rates (G) and suspension densities (Mт) of calcium oxalate (CaOx) crystallization by the mixed suspension mixed product removal (MSMPR) system. Total urinary GAGs, glycoproteins and individual GAGs including heparan sulfate (HS), chondroitin sulfate (CS) and Hyaluronic acid (HA) were added into the artificial urine (AU) and then introduced into the MSMPR test chamber and the crystal sizes and numbers were analyzed by a particle counter. The effects of added GAGs and GPs on CaOx crystallization were reflected by the changes on the crystallization indexes including the Bo, G and Mт of CaOx that were calculated based on the crystal size and numbers. Total urinary GAGs showed no statistical significance on CaOx crystallization. However, individual GAGs such as HA, CS and HS enhanced Bo and suppressed the G when measured individually. CS and HS enhanced the Mт while HA shown no significant change in the Mт of CaOx. Total urinary GPs showed an increase in the G and Mт of crystals. Although total urinary GAGs showed no statistically significant effect on CaOx crystallization, individual GAGs (CS, HS) promoted the CaOx crystallization by increasing the suspension density of smaller crystals, indicative of reduced risk of stones while HA showed no significance in the M(T) of CaOx formed. Urinary GPs indicated increased sizes and M(T) suggesting larger crystals and/or aggregates.     

Glycosaminoglycan polysaccharide biosynthesis and production: today and tomorrow

Glycosaminoglycans [GAGs] are essential heteropolysaccharides in vertebrate tissues that are also, in certain cases, employed as virulence factors by microbes. Hyaluronan [HA], heparin, and chondroitin sulfate [CS] are GAGs currently used in various medical applications and together are multi-billion dollar products thus targets for production by animal-free manufacture. By using bacteria as the source of GAGs, the pathogen’s sword may be converted into a plowshare to help avoid potential liabilities springing from the use of animal-derived GAGs including adventitious agents (e.g., prions, pathogens), antigenicity, degradation of the environment, and depletion of endangered species. HA from microbes, which have a chemical structure identical to human HA, has already been commercialized and sold at the ton-scale. Substantial progress towards microbial heparin and CS has been made, but these vertebrate polymers are more complicated structurally than the unsulfated bacterial polysaccharide precursors thus require additional processing steps. This review provides an overview of GAG structure, medical applications, microbial biosynthesis, and the state of bacterial GAG production systems. Representatives of all glycosyltransferase enzymes that polymerize the sugar chains of the three main GAGs have been identified and serve as the core technology to harness, but the proteins involved in sugar precursor formation and chain export steps of biosynthesis are also essential to the GAG production process. In addition, this review discusses future directions and potential important issues. Overall, this area is poised to make great headway to produce safer (both increased purity and more secure supply chains) non-animal GAG-based therapeutics.     

Analysis of hyaluronan content in chondroitin sulfate preparations by using selective enzymatic digestion and electrospray ionization mass spectrometry

Two important glycosaminoglycans (GAGs) with structural roles in the body's cartilage are hyaluronan (HA) and chondroitin sulfate (CS). A simple mass spectrometric method for determining the amount of HA that may be present in isolated CS samples is presented in this article. Samples are subjected to selective enzymatic digestion using a bacterial hyaluronidase (HA lyase, EC 4.2.2, from Streptococcus dysgalactiae) specific for HA. Undigested CS GAG is then removed by centrifugal filtration, and digested HA left in the filtrate is quantified by electrospray ionization mass spectrometry using an internal standard and selected ion monitoring. The described method was applied to the analysis of several CS samples prepared for use in nutritional supplements.     

花背蟾蜍角膜早期发育中氨基多糖的电镜细胞化学研究

本文用钌红显示氨基多糖的电镜细胞化学方法,较细致地研究了花背蟾蜍(Bufo raddeiStrauch)胚胎发育过程中(16—25期,即神经管至鳃盖完全封闭期)氨基多糖(GAG)在角膜早期发育中的变化。结果表明:GAG的合成由非硫酸化向硫酸化转变,且随着角膜的发育其含量逐渐增加。角膜各部位的HA在16—21期(胚胎开口期),其含量逐渐增加,且20(鳃血循环期)-21期达最高水平,此后含量下降,同时DS、CS、HS和Hep的含量上升。据分析,HA、HS和胶原与间充质细胞迁移有关,HA还可促使基质的膨胀和水化。硫酸化GAG在角膜的脱水、致密、细胞密度及角膜透明中起作用。胶原纤维间的硫酸化GAG能调节胶原纤维的规则化,故也有助于角膜的透明。     

Glycosaminoglycans enhance megakaryocytopoiesis by modifying the activities of hematopoietic growth regulators

We have previously reported that heparin is capable of stimulating in vitro and in vivo megakaryocytopoiesis in mice and has a thrombopoietic effect when given in chronic immune thrombocytopenic purpura and that heparin and several other glycosaminoglycans (GAGs) promote the growth of human megakaryoblastic cell lines in the presence of serum. We show here that GAGs, including heparan sulfate (HS), chondroitin sulfate (CS), dermantan sulfate (DS), and hyaluronic acid (HA), also stimulate in vitro growth of murine megakaryocyte progenitors and augment the diameter of individual megakaryocytes in the presence of serum. However, in a serum-free agar system, the GAGs alone had no effect on megakaryocyte colony formation, suggesting that GAGs cooperate with some serum factor(s) to exert their activity. We also show that heparin significantly potentiates the megakaryocytopoietic activity of C-Mpl ligand and interleukin (IL)-6 but not IL3, GM-CSF, SCF, and Epo. In addition, the GAGs significantly neutralize the inhibitory action of platelet factor 4 (PF4) and transforming growth factor β1 (TGFβ1) on megakaryocyte colony growth. These results demonstrate a stimulating activity of GAGs on megakaryocytopoiesis by modifying the activity of several growth-regulating factors. © 1996 Wiley-Liss, Inc.     

The structural stability of the endothelial glycocalyx after enzymatic removal of glycosaminoglycans

Rationale

It is widely believed that glycosaminoglycans (GAGs) and bound plasma proteins form an interconnected gel-like structure on the surface of endothelial cells (the endothelial glycocalyx layer–EGL) that is stabilized by the interaction of its components. However, the structural organization of GAGs and proteins and the contribution of individual components to the stability of the EGL are largely unknown.

Objective

To evaluate the hypothesis that the interconnected gel-like glycocalyx would collapse when individual GAG components were almost completely removed by a specific enzyme.

Methods and Results

Using confocal microscopy, we observed that the coverage and thickness of heparan sulfate (HS), chondroitin sulfate (CS), hyaluronic acid (HA), and adsorbed albumin were similar, and that the thicknesses of individual GAGs were spatially nonuniform. The individual GAGs were degraded by specific enzymes in a dose-dependent manner, and decreased much more in coverage than in thickness. Removal of HS or HA did not result in cleavage or collapse of any of the remaining components. Simultaneous removal of CS and HA by chondroitinase did not affect HS, but did reduce adsorbed albumin, although the effect was not large.

Conclusion

All GAGs and adsorbed proteins are well inter-mixed within the structure of the EGL, but the GAG components do not interact with one another. The GAG components do provide binding sites for albumin. Our results provide a new view of the organization of the endothelial glycocalyx layer and provide the first demonstration of the interaction between individual GAG components.     

Keratan sulfate epitopes exhibit a conserved distribution during joint development that remains undisclosed on the basis of glycosaminoglycan charge density.

Changes in glycosaminoglycan (GAG) content and distribution are vital for joint development. However, their precise character has not been established. We have used immunohistochemistry (IHC) and "critical electrolyte" Alcian blue staining to assess such changes in developing chick and rabbit joints. IHC showed chondroitin sulfate labeling in chick epiphyseal cartilage but not in interzones. In contrast, prominent labeling for keratan sulfate (KS) was restricted to chick cartilage-interzone interfaces. In rabbit knees, KS labeling was also prominent at presumptive cavity borders, but weak in interzone and cartilage. Selective pre-digestion produced appropriate loss of label and undersulfated KS was undetectable. Quantification of Alcian blue staining by scanning and integrating microdensitometry showed prominent hyaluronan-like (HA-like) interzone staining, with chondroitin sulfate and weaker KS staining restricted to epiphyseal cartilage. Hyaluronidase decreased HA-like staining in the interzone. Surprisingly, keratanases also reduced HA-like but not sulfated GAG (sGAG-like) staining in the interzone. Chondroitinase ABC had little effect on HA-like staining but decreased sGAG staining in all regions. Rabbit joints also showed HA-like but not KS staining in the interzone and strong chondroitin sulfate-like staining in epiphyseal cartilage. Our findings show restricted KS distribution in the region close to the presumptive joint cavity of developing chick and rabbit joints. Alcian blue staining does not detect this moiety. Therefore, it appears that although histochemistry allows relatively insensitive quantitative assessment of GAGs, IHC increases these detection limits. This is particularly evident for KS, which exhibits immunolabeling patterns in joints from different species that is consistent with a conserved functional role in chondrogenesis.     

Characterization of the recombinant rat 175-kDa hyaluronan receptor for endocytosis (HARE)

Hyaluronan (HA) and chondroitin sulfate (CS) clearance from lymph and blood in mammals is mediated by the HA receptor for endocytosis (HARE), which is present as two isoforms in rat and human (175/300 kDa and 190/315 kDa, respectively) in the sinusoidal endothelial cells of liver, spleen, and lymph nodes (Zhou, B., McGary, C. T., Weigel, J. A., Saxena, A., and Weigel, P. H. (2003) Glycobiology 13, 339-349). The small rat and human HARE proteins are not encoded directly by mRNA but are derived from larger precursors. Here we characterize the specificity and function of the 175-kDa HARE, expressed in the absence of the 300-kDa species, in stably transfected SK-Hep-1 cells. The HARE cDNA was fused with a leader sequence to allow correct orientation of the membrane protein. The recombinant rHARE contained approximately 25 kDa of N-linked oligosaccharides and, like the native protein, was able to bind HA in a ligand blot assay, even after de-N-glycosylation. SK-HARE cell lines demonstrated specific 125I-HA endocytosis, receptor recycling, and delivery of HA to lysosomes for degradation. The Kd for the binding of HA (number-average molecular mass approximately 133 kDa) to the 175-kDa HARE at 4 degrees C was 4.1 nm with 160,000 to 220,000 HA-binding sites per cell. The 175-kDa rHARE binds HA, dermatan sulfate, and chondroitin sulfates A, C, D, and E, but not chondroitin, heparin, heparan sulfate, or keratan sulfate. Surprisingly, recognition of glycosaminoglycans (GAGs) other than HA by native or recombinant HARE was temperature-dependent. Although competition was observed at 37 degrees C, none of the other GAGs competed for 125I-HA binding to SK-HARE cells at 4 degrees C. Anti-HARE monoclonal antibody-174 showed a similar temperature-dependence in its ability to block HA endocytosis. These data suggest that temperature-induced conformational changes may alter the GAG specificity of HARE. The results confirm that the 175-kDa rHARE does not require the larger HARE isoform to mediate endocytosis of multiple GAGs.