Binding of human phospholipase A2 type II to proteoglycans. Differential effect of glycosaminoglycans on enzyme activity

Phospholipase A2 acting on low density lipoproteins in the extracellular arterial intima may form proinflammatory lipid mediators. Human nonpancreatic secretory phospholipase A2 has three regions that may associate with sulfated glycosaminoglycans. The apoB-100 molecule in low density lipoproteins also has glycosaminoglycan binding regions that could mediate its retention in the arterial intima. Here we report that human nonpancreatic phospholipase A2 isolated from a transfected cell line binds to glycosaminoglycans secreted by cultured human arterial smooth muscle cells. A gel mobility shift assay showed that the affinity of phospholipase A2 for glycosaminoglycans from a heparan sulfate/chondroitin sulfate proteoglycan was higher than for chondroitin sulfate glycosaminoglycans from a larger versican-like proteoglycan. Affinity chromatography confirmed these results. All glycosaminoglycans tested, at concentrations up to 100 microM, increased the activity of phospholipase A2 toward phosphatidylcholine liposomes. Above this concentration, heparan sulfate and heparin inhibited the enzyme. Heparin and chondroitin 6-sulfate increased phospholipase A2 activity on low density lipoproteins up to 4-fold at 100 microM, whereas heparan sulfate had no effect. The results indicate that human nonpancreatic secretory phospholipase A2 interacts with proteoglycans via their glycosaminoglycan moiety and that the enzyme activity may be modulated by the association of the enzyme and its substrate to the sulfated polysaccharides.     

Developmental distribution of glycosaminoglycans in embryonic rat brain: relationship to axonal tract formation

Glycosaminoglycans, the sugar moieties of proteoglycans, modulate axonal growth in vitro. However, their anatomical distribution in relation to developing axonal tracts in the rat brain has not been studied. Here, we examined the immunohistochemical distribution of chondroitin-6-sulfate and chondroitin-4-sulfate, two related glycosaminoglycan epitopes, which are present in three types of glycosaminoglycans: chondroitin sulfate C, chondroitin sulfate A, and chondroitin sulfate B. Further, we compared their distribution pattern to that of axonal tract development. Both glycosaminoglycan epitopes showed a heterogeneous spatiotemporal distribution within the developing rat brain. However, the expression of chondroitin-4-sulfate was more restricted than that of chondroitin-6-sulfate, although both epitopes were detected from embryonic day 13 until the day of birth, overlapping in many regions of the central nervous system including cortex, hippocampus, thalamus, and hindbrain. After birth, the levels of expression of both glycosaminoglycan epitopes progressively decreased and were practically undetectable after the first postnatal week. The expression of chondroitin-6-sulfate and, to a lesser extent, that of chondroitin-4-sulfate, was preferentially associated to the extracellular matrix surrounding specific axon bundles. However, the converse association was not true, and several apparently similar types of axon developed on a substrate devoid of both types of glycosaminoglycan epitopes. These results provide an anatomical background for the idea that different types of glycosaminoglycans may contribute to establish the complex set of guidance cues necessary for the specific development of defined axon tracts in the central nervous system.     

Binding of low density lipoproteins by proteoglycans synthesized by proliferating and quiescent human arterial smooth muscle cells

Chondroitin sulfate-rich proteoglycans secreted by arterial intima smooth muscle cells appear involved in low density lipoprotein entrapment and modification. Hypothetically, such a process may contribute to atherogenesis. We compared composition and size of those proteoglycans synthesized by proliferating and resting human arterial smooth muscle cells for which low density lipoprotein had affinity. Lipoprotein-binding proteoglycans secreted by proliferating cells were larger than those of resting cells (M(r) = 1.1 x 10(6) versus 0.8 x 10(6). This was primarily caused by increased M(r) of the chondroitin sulfate chains (6 x 10(4) versus 3.5 x 10(4)). The glycosaminoglycan chains of the proteoglycans from both cells were made of more than 90% chondroitin 6-sulfate and chondroitin 4-sulfate in a 6:4 ratio. Affinity chromatography indicated that low density lipoprotein had a higher affinity with the proteoglycans synthesized by proliferating cells than those from resting cells. Measured with gel mobility shift assay, the apparent affinity constant of low density lipoproteins for proteoglycans from proliferating cells was 3-fold higher than that for proteoglycans from resting cells. This increased affinity appeared related to the higher relative proportion of proteoglycans with longer glycosaminoglycan chains secreted by the proliferating cells than those secreted by the resting cells.     

Glycosaminoglycan and collagen distribution in the developing human vitreous

BACKGROUND: We determined the distribution of glycosaminoglycans and collagens in the developing human vitreous. METHODS: Eighty human eyes from 5 gestational weeks to 2 postnatal years of age were used. Glycosaminoglycan components were determined by enzyme digestion with hyaluronidase or chondroitinase AC and ABC and immunohistochemistry for chondroitin, chondroitin-4-sulfate, chondroitin-6-sulfate, and dermatan sulfate. Collagen distribution was determined by immunohistochemistry for types I, II, and III collagens. RESULTS: Enzyme digestion showed that throughout development hyaluronic acid is the main glycosaminoglycan in the vitreous and in the extraocular space at 5-7 gestational weeks. Both areas were filled with mesenchymal cells. Immunohistochemistry showed chondroitin-6-sulfate in the vitreous between 6 and 40 gestational weeks, and chondroitin-4-sulfate between 12 and 40 gestational weeks. Hyaluronic acid and chondroitin sulfate appeared in the retina and around the hyaloid vessels at 12-40 weeks. Immunohistochemistry showed type III collagen in the vitreous and around the mesenchymal cells at 5-7 weeks that was replaced by type II collagen after 8 weeks. CONCLUSIONS: Hyaluronic acid is the major glycosaminoglycan in the vitreous throughout development, except for the transient appearance of chondroitin sulfate at 6-40 gestational weeks. Type III is the main collagen in the early developing vitreous that converts to type II collagen at 8 weeks. The primary and secondary vitreous has the same components as these macromolecules. These vitreous glycosamino-glycans and collagens seem to be produced by mesenchymal cells at an early stage and by the retina and hyaloid vessels during middle and late development.     

Age-related changes in populations of aortic glycosaminoglycans: species with low affinity for plasma low-density lipoproteins, and not species with high affinity, are preferentially affected

Glycosaminoglycans were extracted from the intima and media layers of normal human thoracic aortas from donors of different ages. The arterial segments were devoid of macroscopically visible lesions obtained from patients who had no clinically evident cardiovascular disease. Total glycosaminoglycan content increases during the first 40 years of life. Changes in the content of hyaluronic acid and heparan sulfate are less noticeable. The content of chondroitin sulfate (mainly the 6-isomer) increases, whereas dermatan sulfate remains constant. Plasma LDL-affinity chromatography of dermatan sulfate+chondroitin 4/6-sulfate fractions allowed the separation of LDL high- and low-affinity glycosaminoglycan species. Remarkably, only glycosaminoglycan species with low affinity for plasma LDL increase with age in the disease-free areas of human thoracic aortas studied. These results suggest that age-related changes in glycosaminoglycan composition of the arterial wall do not contribute to increased deposition of plasma LDL. However, the alternative explanation that individuals with arterial glycosaminoglycans that avidly bind LDL would develop early and severe cardiovascular disease and would thus be excluded from our analysis cannot be ruled out.     

Purification and properties of human N-acetylgalactosamine-6-sulfate sulfatase

1. Human N-acetylgalactosamine-6-sulfate sulfatase (EC 3.1.6.-) from human placenta has been purified more than 3000-fold by gel filtration, ion-exchange and substrate affinity chromatography. The enzyme has a molecular weight of 90 000 by gel filtration chromatography and 85 000 by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Enzyme purified from cultured human skin fibroblasts has similar properties. 2. The tritium-labeled chrondroitin 6-sulfate trisaccharide N-acetylgalactosamine 6-sulfate-(beta, 1-4)-glucuronic acid-(beta, 1-3(-N-acetyl[1-3H]galactosaminitol 6-sulfate as substrate demonstrated a Km of 0.12 mM at pH 4.5. Sulfate was hydrolyzed only from the non-reducing terminal of this disulfated trisaccharide. Hyaluronic acid, dermatan sulfate, chondroitin 4-sulfate, heparin and chondroitin 6-sulfate tetrasaccharide were slightly inhibitory, whereas 6-sulfated pentasaccharides and heptasaccharides were strongly inhibitory. The enzyme dose not hydrolyze sulfate from N-acetylglucosamine 6-sulfate.     

Widespread expression of chondroitin sulfate-type serglycins with CD44 binding ability in hematopoietic cells

Serglycin is a family of small proteoglycans with Ser-Gly dipeptide repeats and is modified with various types of glycosaminoglycan side chains. We previously demonstrated that chondroitin sulfate-modified serglycin is a novel ligand for CD44 involved in the adherence and activation of lymphoid cells. In this study, we investigated the production and distribution of CD44 binding serglycins in various hematopoietic cells and characterized their carbohydrate side chains. Immunoprecipitation analysis using CD44-IgG and polyclonal antibody against the serglycin core peptide demonstrated that various serglycin species capable of binding CD44 are produced by a variety of hematopoietic cells including lymphoid cells, myeloid cells, and a few tumor cell lines. Glycosaminoglycans on these serglycins, which are essential for CD44 binding, are composed of chondroitin 4-sulfate or a mixture of chondroitin 4-sulfate and chondroitin 6-sulfate, but no heparin or heparan sulfate side chain was detected. The serglycins are also secreted by normal splenocytes, lymph node lymphocytes, and bone marrow cells, whereas they are secreted in very small amounts by normal thymocytes. Secretion of serglycins is greatly enhanced by mitogenic stimulation with concanavalin A or lipopolysaccharide. Our results showed that serglycin, unlike hyaluronate, is produced and secreted in a functional (CD44 binding) form by many members of the hematopoietic system including various lymphocyte subsets. Our data suggest that serglycin may serve as a major ligand for CD44 in various events in the lymphohematopoietic system.     

Modulation of cAMP-dependent protein kinase by derivatives of chondroitin sulfate

Here, we report investigations about the direct effect of glycosaminoglycans, such as dermatan sulfate, chondroitin 4- and 6-sulfate upon cAMP-dependent protein kinase activity. The results indicate that glycosaminoglycans strongly influence the phosphorylation activity of this enzyme against histone type IIa and [Val6, Ala7]-kemptide. While chondroitin 4-sulfate and dermatan sulfate exhibit inhibitory effects, chondroitin 6-sulfate shows a stimulating effect. In addition, the chondroitin 6-sulfate is also able to reduce the chondroitin 4-sulfate and dermatan sulfate specific inhibition.     

Inhibition of human leukocyte elastase by chemically and naturally oversulfated galactosaminoglycans

Several samples of oversulfated chondroitin and dermatan were obtained by chemical sulfation and by SAX-HPLC enrichment. The starting products and oversulfated products were tested as potential inhibitors of human leukocyte elastase, an enzyme hypothesized to be involved in the etiology of diseases such as emphysema, atherosclerosis, and rheumatoid arthritis. Chemical oversulfation (SO3H/COOH 1.6-3.2), preferentially occurring at C-6 of galactosamine residues, was found generally to increase the inhibitory power on elastase. Chemically oversulfated galactosaminoglycans thus have potential as therapeutic agents, considering that they produce non-significant effects on the hemocoagulative system. Two naturally oversulfated dermatans sulfate (SO3H/COOH ca. 1.2), mainly oversulfated at C-2 of iduronic acid residues, showed comparatively higher anticoagulant activity (in the HC-II mediated thrombin inhibition test).     

SPACR, a novel interphotoreceptor matrix glycoprotein in human retina that interacts with hyaluronan

SPACR (sialoprotein associated with cones and rods), is the major 147-150-kDa glycoprotein present in the insoluble interphotoreceptor matrix of the human retina. Immunocytochemistry localizes SPACR to the matrix surrounding rods and cones (Acharya, S., Rayborn, M. E., and Hollyfield, J. G. (1998) Glycobiology 8, 997-1006). From affinity-purified SPACR, we obtained seven peptide sequences showing 100% identity to the deduced sequence of IMPG1, a purported chondroitin 6-sulfate proteoglycan core protein, which binds peanut agglutinin and is localized to the interphotoreceptor matrix. We show here that SPACR is the most prominent 147-150-kDa band present in the interphotoreceptor matrix and is the gene product of IMPG1. SPACR is not a chondroitin sulfate proteoglycan, since it is not a product of chondroitinase ABC digestion and does not react to a specific antibody for chondroitin 6-sulfate proteoglycan. Moreover, the deduced amino acid sequence reveals no established glycosaminoglycan attachment site. One hyaluronan binding motif is present in the predicted sequence of SPACR. We present evidence that SPACR has a functional hyaluronan binding domain, suggesting that interactions between SPACR and hyaluronan may serve to form the basic macromolecular scaffold, which comprises the insoluble interphotoreceptor matrix.     

Hypereosinophilic syndrome in a child mosaic for a congenital triplication of the short arm of chromosome 8

Fibroblast growth factors are important heparin binding, mitogenic proteins. The binding site in heparin and heparan sulfate for fibroblast growth factor-2 (basic fibroblast growth factor) has been described as rich in glucosamine-2-sulfate 1-->4 linked to iduronic acid-2-sulfate. The glucosamine residue in the heparin binding site is also 6-sulfated. A new glycosaminoglycan, acharan sulfate, has been chemically modified to prepare a polysaccharide, N-sulfoacharan sulfate, consisting of glucosamine-2-sulfate 1-->4 linked to iduronic acid-2-sulfate. Acharan sulfate binds very weakly to fibroblast growth factor-2 while N-sulfoacharan sulfate binds with nearly the same affinity as heparin. Mitogenicity studies were performed using heparan sulfate-free cells stably transfected with fibroblast growth factor receptor-1. Acharan sulfate inhibits heparin's enhancement of fibroblast growth factor-2 mitogenic activity, without affecting cell viability, while N-sulfoacharan sulfate shows heparin-like activity but at a greatly reduced level. These results suggest additional mechanisms not requiring high affinity glycosaminoglycan binding to fibroblast growth factor-2 may be important in its mitogenic activity.     

Human monocyte-derived macrophages secrete two forms of proteoglycan-macrophage colony-stimulating factor that differ in their ability to bind low density lipoproteins

This study evaluated whether human monocyte-derived macrophages synthesize specific types of proteoglycans with lipoprotein-binding capability that could contribute to lipid retention in the arterial wall. After labeling with either [35S]SO4 or [35S]methionine, macrophages secreted a high molecular mass proteoglycan, with glycosaminoglycan chains of approximately 18 kDa and core protein bands of approximately 100 and 55 kDa. Both core protein bands were recognized by an antibody to PG-100, an antibody that recognizes the proteoglycan form of macrophage colony-stimulating factor (PG-100/PG-MCSF). The interaction between PG-100/PG-MCSF and low density lipoproteins (LDL) was examined by gel mobility shift. In this system, PG-100/PG-MCSF was resolved further into two forms. The two forms had the same core proteins but differed in their overall size and glycosaminoglycan content. The larger form contained glycosaminoglycan chains that were entirely chondroitin ABC lyase-sensitive, whereas the smaller form contained chains that were sensitive to both chondroitin ABC lyase and heparinase. Both forms bound native LDL with high affinity, but the larger form bound LDL with higher affinity than the smaller form. The glycosaminoglycan chains of PG-100/PG-MCSF, but not the core proteins, were responsible for binding to native LDL. Mildly oxidized LDL and methyl-LDL, which have an electrophoretic charge similar to that of native LDL, also bound PG-100/PG-MCSF. In contrast, extensively oxidized LDL and acetyl-LDL, which are more electronegative than native LDL, did not bind to either form of PG-100/PG-MCSF. The demonstration of two forms of human monocyte-derived macrophage PG-100/PG-MCSF which bind LDL may represent an additional role for macrophages in the extracellular trapping of lipoproteins in atherosclerosis.     

Successful treatment of intraoperative myocardial ischemia with nicorandil

The relationship between sulfation and polymerization in chondroitin sulfate (CS) biosynthesis has been poorly understood. In this study, we investigated the specificity of bovine serum UDP-GalNAc: CS beta-GalNAc transferase responsible for chain elongation using structurally defined acceptor substrates. They consisted of tetra- and hexasaccharide-serines that were chemically synthesized and various regular oligosaccharides with a GlcA residue at the nonreducing terminus, prepared from chondroitin and CS using testicular hyaluronidase. The enzyme preparation was obtained from fetal bovine serum by means of heparin-Sepharose affinity chromatography. The preparation did not contain the alpha-GalNAc transferase recently demonstrated in fetal bovine serum (Kitagawa et al., J. Biol. Chem., 270, 22190-22195, 1995), that utilizes common acceptor substrates. The beta-GalNAc transferase used as acceptors, two hexasaccharide-serines GlcA beta 1-3GalNAc beta 1-4GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser and GlcA beta 1-3GalNAc(4-sulfate) beta 1-4GlcA beta 1-3Gal (4-sulfate) beta 1-3Gal beta 1-4Xyl beta 1-O-Ser, but neither the monosulfated hexasaccharide-serine GlcA beta 1-3GalNAc(4-sulfate) beta 1-4GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser nor tetrasaccharide-serines with or without a sulfate group at C-4 of the third sugar residue Gal-3 from the reducing end. The results indicated that the sulfate group at the Gal-3 C-4 markedly affected the transfer of GalNAc to the terminal GlcA. In addition, a sulfate group at C-4 of the reducing terminal GalNAc of regular tetrasaccharides remarkably enhanced the GalNAc transfer, suggesting that the enzyme recognizes up to the fourth saccharide residue from the nonreducing end. The level of incorporation into a tetra- or hexasaccharide containing a terminal 2-O-sulfated GlcA residue was significant, whereas there was no apparent incorporation into tetra- or hexasaccharides containing a terminal 3-O-sulfated GlcA or penultimate 4,6-O-disulfated GalNAc residue. These results indicated that sulfation reactions play important roles in chain elongation and termination.     

A sensitive enzymatic-isotopic method for the analysis of tyramine in brain and other tissues

The concentration, composition and sulfate labeling of glycosaminoglycans and glycoproteins have been studied in purified nuclei isolated in bulk from rat brain. The concentration of total glycosaminoglycans is 0.142 mumol hexosamine/100 mg protein, comprising 57% chondroitin 4-sulfate, 7% chondroitin 6-sulfate, 29% hyaluronic acid and 7% heparan sulfate. Control experiments demonstrated that less than 5% of the sulfated glycosaminoglycans associated with nuclei could be accounted for by the nonspecific adsorption of soluble acidic proteoglycans to basic nuclear proteins. Glycoprotein carbohydrate is present at a level of 206 mug/100 mg protein, and has an average composition of 30% N-acetylglucosamine, 29% mannose, 19% N-acetylneuraminic acid, 15% galactose, 4% N-acetylgalactosamine, and 3% fucose. Labeling studies also indicated the presence of ester sulfate residues on the glycoprotein oligosaccharides.     

A role of chondroitin sulfate glycosaminoglycan binding site in alpha4beta1 integrin-mediated melanoma cell adhesion

We have previously reported that alpha4beta1 (but not alpha5beta1) integrin-mediated melanoma cell adhesion is inhibited by removal of cell surface chondroitin sulfate glycosaminoglycan (CSGAG), suggesting that melanoma chondroitin sulfate proteoglycan plays a role in modulating the adhesive function of alpha4beta1 integrin. In the current study, we demonstrated that alpha4beta1 integrin binds to CSGAG. We have identified a peptide from within alpha4 integrin termed SG1 (KKEKDIMKKTI) that binds to cell surface melanoma chondroitin sulfate proteoglycan, indicating that SG1 represents a CSGAG binding site within the alpha4 integrin subunit. Soluble SG1 inhibits alpha4beta1 integrin-mediated human melanoma cell adhesion to CS1. Polyclonal antibody generated against the peptide inhibits melanoma cell adhesion to CS1, and the inhibition is reversed by Mn2+ and an activating monoclonal antibody anti-beta1 (8A2). Additionally, pretreatment of cells with anti-SG1 IgG inhibits the expression of the monoclonal antibody 15/7 epitope in the presence of soluble CS1 peptide, suggesting that anti-SG1 IgG prevents ligand binding by alpha4beta1 integrin. These results demonstrate that alpha4beta1 integrin interacts directly with CSGAG through SG1 site, and that this site can affect the ligand binding properties of the integrin.     

Model calculation of the crystal-field magnetostriction and its temperature dependence in the itinerant uniaxial ferromagnet Y2Fe17

A stimulatory effect of increased salt content on the metabolism of benzphetamine, 7-ethoxycoumarin, and coumarin by rabbit liver microsomes, CYP2B4 and rabbit CYP1A2, was seen, indicating that the effect was not specific for either substrate or form of cytochrome P450. The stimulation was not due to an action on the cytochrome P450 itself as increased salt concentration minimally affected the substrate turnover when cumene hydroperoxide was used as the source of active oxygen. The elevation of ionic strength increased the coupling efficiency of the monooxygenase reaction with benzphetamine as substrate. Cytochrome b5 also can increase the monooxygenase coupling efficiency. At low ionic strength cytochrome b5 did not much influence the reduction of P450, but the rate constant of the cytochrome b5 reduction was increased about 15-fold by its binding to cytochrome P450. A stimulatory effect of cytochrome b5 on benzphetamine oxidation was seen at low ionic strength, but it was lost at elevated ionic strength as the binding of cytochrome b5 to cytochrome P450 was weakened. At the higher ionic strength cytochrome b5 competes with cytochrome P450 for the reductase, an action that slows cytochrome P450 reduction. Based upon these observations, plus those in the literature, a scheme is suggested that proposes the stimulatory effect of cytochrome b5 on the cytochrome P450-mediated monooxygenation reaction is due to an increase in the efficiency of the electron transfer reaction: With cytochrome b5 bound to cytochrome P450, two electrons can be provided from the reductase to the P450-b5 complex in a single interaction, obviating the need for a second interaction with the reductase.     

Bikunin present in human peritoneal fluid is in part derived from the interaction of serum with peritoneal mesothelial cells

We recently reported that peritoneal fluid mainly contains two proteoglycans; one is the interstitial proteoglycan referred to as decorin, and the other an uncharacterized small chondroitin sulfate proteoglycan. In the present study, we have used a two-step process to isolate the small chondroitin sulfate proteoglycan free of decorin. The purified molecule ran as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with apparent molecular mass 50 kd made up of a chondroitin-4-sulfate glycosaminoglycan chain and a 30-kd core protein. NH2-terminal analysis of the core protein showed significant sequence homology with bikunin, a component of the human inter-alpha-trypsin inhibitor (IalphaI) family. A Western blot analysis using anti-human inter-alpha-trypsin inhibitor confirmed the identity of the small chondroitin sulfate proteoglycan as bikunin, and a trypsin inhibitor counterstain assay confirmed its anti-trypsin activity. Examination of serum from patients receiving continuous peritoneal dialysis suggests that free bikunin in peritoneal fluid may be the result of leakage of serum proteins into the peritoneum. Our findings further show that the interaction of serum with peritoneal mesothelial cells offers a new and novel explanation for the presence of bikunin in peritoneal fluid.     

Differential release of proteoglycans during human B lymphocyte maturation

Proteoglycans interact with soluble proteins such as growth factors and thereby regulate extracellular signals. During B lymphocyte maturation, secretion of proteoglycans may be functionally related to the different requirements of the respective maturation stage. In order to address this question we compared structures of proteoglycans released by three B lymphocyte lines which correspond to different maturation stages. Plasma-cell type U266 cells secreted the largest proteoglycans (150 kDa), followed by mature B cells JOK-1 (130 kDa) and pre-B cells Nalm 6 (90 kDa). On average, secreted proteoglycans carried four glycosaminoglycan chains with molecular masses ranging each from 32 kDa (U266) to 23 kDa (Nalm 6). All three cell lines secreted more than 90% of their proteoglycans possessing chondroitin sulfate chains having chondroitin-4-sulfate (delta Di-4S) as the prevalent disaccharide unit. In these proteochondroitin sulfates, unsulfated chondroitin (delta Di-0S) was present in smaller quantities and chondroitin-6-sulfate (delta Di-6S)-containing proteoglycan was released only by Nalm 6 and U266 cells. Cell line Nalm 6 exclusively produced proteochondroitin sulfate, whereas in culture medium of JOK-1 and U266 a small amount of proteoheparan sulfate was found also. In all three cell lines, treatment with chondroitinase ABC released a protein of 30 kDa and chemical deglycosylation resulted in a core protein of 21 kDa. In addition to pure proteochondroitin sulfate, a small portion of proteoheparan sulfate with a protein moiety of 30 kDa was detected after heparitinase treatment in supernatants of JOK-1 and U266. Thus, our results indicate that released proteoglycans may undergo modulations in their glycosaminoglycan moieties during B-cell differentiation. This may have functional consequences at the level of growth factor regulation.     

Specificity and reactivity of human leukocyte elastase, porcine pancreatic elastase, human granulocyte cathepsin G, and bovine pancreatic chymotrypsin with arylsulfonyl fluorides. Discovery of a new series of potent and specific irreversible elastase inhibitors

The reactivity and specificity of a series of substituted benzenesulfonyl fluorides with human leukocyte (HL) elastase, cathepsin G, porcine pancreatic (PP) elastase, and bovine chymotrypsin A alpha are reported. Benzenesulfonyl fluorides with 2-fluoroacyl substituents were found to be potent and specific inhibitors of elastase. HL elastase was inhibited most rapidly by 2--(CF3CF2CONH)--C6H4SO4F (kobs/[I] = 1700 M-1 s-1) which is slightly better than our best peptide chloromethyl ketone inhibitor of this enzyme. PP elastase was most rapidly inhibited by 2--(CF3CONH)--C6H4-SO2F (kobs/[I] = 2300 M-1S-1). The 2--(CF3CF2CF2CONH) and 2--(CF3SNH) derivatives were quite selective for HL elastase and inhibited PP elastase, cathepsin G, and chymotrypsin A alpha quite slowly. A specific and potent chymotrypsin inhibitor (2--(Z--Gly--NH)--C6H4SO2F) was also discovered. A model for the elastase inhibition reaction is proposed which involves interaction of the fluroacyl group of the inhibitor with the primary substrate recognition site S1 of the enzyme. Hydrogen bonding also occurs between the inhibitor NH and a backbone peptide carbonyl group, probably from residue 214. The 2-fluoroacyl group plays the dual role of binding in the hydrophobic S1 pocket and through electronic effects, increasing the strength f the hydrogen bond. The results of this study demonstrate that it is possible to construct simple organic molecules which are specific inhibitors of HL elastase, PP elastase, or chymotrypsin.     

Two-year prospective, randomized trial comparing an innovative twice-a-week progestin regimen with a continuous combined regimen as postmenopausal hormone therapy

OBJECTIVE: To determine the relationship between matrix metalloproteinases (MMPs), their inhibitors, and the turnover of matrix molecules in articular cartilage from patients with osteoarthritis (OA). METHODS: Synovial fluid samples were collected from the knees of 54 patients with OA. Radiographic evaluations and magnetic resonance imaging were performed on the knees of 34 OA patients to classify the stage of the disease. Biochemical analyses and immunoassays were used to measure the concentrations of MMP-1, MMP-3, tissue inhibitor of metalloproteinases 1 (TIMP-1), TIMP-2, the disaccharide of hyaluronic acid, the proteoglycan glycosaminoglycan disaccharides of chondroitin 4-sulfate (delta di-CS4) and chondroitin 6-sulfate (delta di-CS6), the 846 epitope on chondroitin sulfate of cartilage proteoglycan aggrecan (putative biosynthetic marker), the keratan sulfate (KS) epitope of aggrecan (putative degradation marker), and the C-propeptide of cartilage type II procollagen (CPII) (biosynthetic marker). RESULTS: The concentration of TIMP-1 was directly correlated with the levels of MMP-1 and MMP-3 (both were also correlated with each other), confirming earlier results. There was an inverse correlation between the delta di-CS6:delta di-CS4 ratio and the concentration of MMP-3. The level of delta di-CS6 was correlated with that of the KS epitope, and to a lesser degree, with that of the 846 epitope (the latter was also correlated with the level of delta di-CS4). The concentration of TIMP-1 correlated with that of the 846 epitope, whereas TIMP-2 levels correlated with those of CPII. There were significantly lower concentrations of delta di-CS6, delta di-CS4, the 846 epitope, and CPII in synovial fluid from patients with late-stage OA. CONCLUSION: These observations suggest a link between proteolysis and inhibitor concentrations in OA cartilage. Production of TIMPs appears to be individually linked to the synthesis of specific cartilage molecules. The reduction in the amount of cartilage-matrix structural components suggests that there is a measurable loss of cartilage in the late stages of the disease, as suggested previously. The resultant composition of the cartilage suggests that the loss may primarily involve "resident" molecules originally present in healthy cartilage.