Isoforms of corneal keratan sulfate proteoglycan

Bovine corneal keratan sulfate proteoglycan was found to contain three major protein components. Two proteins (37 and 25 kDa) were released from the proteoglycan by endo-beta-galactosidase, N-glycanase, or chemical deglycosylation. A smaller protein (20 kDa), not covalently linked to keratan sulfate, co-purified with the proteoglycan by conventional and high performance ion exchange chromatography, by ethanol precipitation, and by affinity purification on columns of monoclonal antibody to keratan sulfate, but could be separated from the proteoglycan by gel filtration chromatography in dissociative agents. The three proteins produced different fragmentation patterns on sodium dodecyl sulfate-polyacrylamide gel electrophoresis after digestion with V8 protease, and each had unique two-dimensional tryptic peptide maps. The N-terminal amino acid sequence of the core proteins differed. In addition, the proteoglycans containing these proteins differed in molecular size, suggesting different levels of glycosylation of the two core proteins. Similarity of the core proteins was suggested by similar amino acid composition, similarities in tryptic maps, and antigenic cross-reactivity. Corneal keratan sulfate proteoglycan, therefore, seems to occur in two different, but related, forms whose core proteins may represent members of a homologous family.     

Enzyme-linked immunosorbent assay for hyaluronate using cartilage proteoglycan and an antibody to keratan sulfate

An enzyme-linked immunosorbent assay has been developed to measure hyaluronate concentrations in biological samples. The assay is based on the aggregation of hyaluronate with cartilage proteoglycan monomer, followed by binding of a monoclonal antibody to the keratan sulfate on the proteoglycan. The sensitivity of the assay is 10 ng hyaluronate/ml of either serum or conditioned cell culture medium. The coefficient of variability was between 10 and 20%. Hyaluronate added to samples was recovered quantitatively and digestion of cell culture medium with protease did not affect the concentration of hyaluronate. Hyaluronate polysaccharides as small as a decasaccharide can be measured. This sensitive and convenient assay can be used for measuring large numbers of biological samples from a variety of animal and tissue sources.     

Distribution of proteoglycans antigenically related to corneal keratan sulfate proteoglycan

Three antibodies reacting with corneal keratan sulfate proteoglycan were used to detect antigenically related molecules in 11 bovine and 13 embryonic chick tissues. Two monoclonal antibodies recognized sulfated epitopes on the keratan sulfate chain and a polyclonal antibody bound antigenic sites on the core protein of corneal keratan sulfate proteoglycan. Competitive immunoassay detected core protein and keratan sulfate antigens in guanidine HCl extracts of most tissues. Keratan sulfate antigens of most bovine tissues were only partially extracted with guanidine HCl, but the remainder could be solubilized by CNBr treatment of the guanidine-extracted residue. Keratan sulfate and core protein antigens co-eluted with purified corneal keratan sulfate proteoglycan on ion exchange high-performance liquid chromatography (HPLC). Endo-beta-galactosidase digestion of the HPLC-purified keratan sulfate antigens eliminated the binding of monoclonal anti-keratan sulfate antibodies in enzyme-linked immunosorbent assay. Extracts of all 11 bovine tissues, except those from brain and cartilage, could bind both anti-keratan sulfate monoclonal antibodies and anti-core protein polyclonal antibody simultaneously. Binding was sensitive to competition with keratan sulfate and to digestion with endo-beta-galactosidase. These results suggest widespread occurrence of a proteoglycan or sulfated glycoprotein bearing keratan sulfate-like carbohydrate and a core protein resembling that of corneal keratan sulfate proteoglycan.     

A monoclonal antibody that specifically recognizes a glucuronic acid 2-sulfate-containing determinant in intact chondroitin sulfate chain

Monoclonal antibodies produced against chick embryo limb bud proteoglycan (PG-M) were selected for their ability to recognize determinants on intact chondroitin sulfate chains. One of these monoclonal antibodies (IgM; designated MO-225) reacts with PG-M, chick embryo cartilage proteoglycans (PG-H, PG-Lb, and PG-Lt), and bovine nasal cartilage proteoglycan, but not with Swarm rat chondrosarcoma proteoglycan. The reactivity of PG-H to MO-225 is not affected by keratanase digestion but is completely abolished after chondroitinase digestion. Competitive binding analyses with various glycosaminoglycan samples indicate that the determinant recognized by MO-225 resides in a D-glucuronic acid 2-sulfate(beta 1----3)N-acetylgalactosamine 6-sulfate disaccharide unit (D-unit) common to antigenic chondroitin sulfates. A tetrasaccharide trisulfate containing D-unit at the reducing end is the smallest chondroitin sulfate fragment that can inhibit the binding of the antibody to PG-H. Decreasing the size of a D-unit-rich chondroitin sulfate by hyaluronidase digestion results in progressive reduction in its inhibitory activity. The results suggest that the epitope has a requirement for a long stretch of a disaccharide-repeating structure for a better fit to the antibody.     

Identification of a developmentally regulated keratan sulfate proteoglycan that inhibits cell adhesion and neurite outgrowth.

Monoclonal antibodies have been used to identify a 320 kd keratan sulfate proteoglycan that is primarily expressed in the embryonic chick nervous system. Immunohistochemical localization of the proteoglycan shows that it is expressed by putative midline barrier structures in the developing chick central nervous system. When added to laminin or neural cell adhesion molecule that has been adsorbed onto nitrocellulose-coated dishes, the proteoglycan abolishes cell attachment and neurite outgrowth on these adhesive substrata. This effect can be reversed by keratanase treatment and incubation with a monoclonal antibody that recognizes the keratan sulfate chains of the proteoglycan. These data suggest that this neural keratan sulfate proteoglycan plays an important role in the modulation of neuronal cell adhesion during embryonic brain development.     

Identification of chick corneal keratan sulfate proteoglycan precursor protein in whole corneas and in cultured corneal fibroblasts.

The precursor protein to the chick corneal keratan sulfate proteoglycan was identified by immunoprecipitation with antiserum to its core protein from lysates of [35S]methionine-pulsed corneas and corneal fibroblasts in cell culture. Antiserum to the keratan sulfate proteoglycan immunoprecipitated a doublet of Mr 52,000 and 50,000 and minor amounts of a Mr 40,000 protein from pulsed corneas. Pulse-chase experiments, which permitted the conversion of the precursor proteins to proteoglycans and digestion of the glycosaminoglycans on immunoprecipitated proteoglycans with keratanase or chondroitinase ABC, showed that the Mr 52,000-50,000 doublet was converted to a keratan sulfate proteoglycan and the Mr 40,000 protein was converted to a chondroitin sulfate proteoglycan. Chick corneal fibroblasts in cell culture primarily produced the smaller (Mr50,000) precursor protein, and in the presence of tunicamycin the precursor protein size was reduced to Mr35,000, which indicates that the core protein contains approximately five N-linked oligosaccharides. Pulse-chase experiments with corneal fibroblasts in culture showed that the precursor protein was processed and secreted into the medium. However, its sensitivity to endo-beta-galactosidase and resistance to keratanase indicate that the precursor protein was converted to a glycoprotein with large oligosaccharides and not to a proteoglycan. This suggests that, although the precursor protein for the proteoglycan is produced in cultured corneal fibroblasts, the sulfation enzymes for keratan sulfate may be absent.     

Cell-free translation and characterization of corneal keratan sulfate proteoglycan core proteins.

Bovine corneal keratan sulfate proteoglycan (KSPG) contains two core proteins, 37 and 25 kDa, if fully deglycosylated, but 47 and 35 kDa, respectively, after endo-beta-galactosidase (Funderburgh, J. L., and Conrad, G. W. (1990) J. Biol Chem. 265, 8297-8303). Chicken corneal KSPG released a single core protein of 47 kDa after endo-beta-galactosidase, and of 35 and 36 kDa, if deglycosylated with N-glycanase or trifluoromethanesulfonic acid. Affinity purified rabbit antibodies against each KSPG recognized only the intact proteoglycan or its core proteins in immunoblots of unfractionated guanidine-HCl extracts of whole cornea after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Affinity purified antibody to a synthetic peptide duplicating the NH2-terminal sequence of the 37-kDa bovine core protein showed little reactivity with untreated corneal extract but reacted with the 47-kDa bovine protein in endo-beta-galactosidase-treated extracts. RNA was isolated from bovine and chick corneal stromas and used for in vitro translation. Antibody against bovine KSPG immunoprecipitated two proteins of 56-53 kDa and a protein of 41 kDa after translation of bovine RNA. Translation of chick RNA produced a double band of 38-39 kDa and a single band of 25 kDa precipitating with antibody against chicken KSPG. Homologous unlabeled KSPG competed for binding of antibodies to these translation products. These data suggest that in vertebrate corneas, the multiple KSPG core protein isoforms may arise as products of separate mRNAs, rather than from proteolytic processing of a large polypeptide precursor.     

Studies of a monoclonal antibody to skeletal keratan sulphate. Importance of antibody valency.

A mouse monoclonal antibody (AN9P1) to keratan sulphate is described. In a competitive-inhibition solution-phase radioimmunoassay employing 125I-labelled intact proteoglycan, it reacts preferentially with keratan sulphate bound to the core protein of adult human articular-cartilage proteoglycan and to a much lesser degree with keratan sulphate purified from this proteoglycan. Proteolytic cleavage of the proteoglycan by pepsin and trypsin has little effect on antibody binding, but treatment with papain decreases binding considerably and more than does treatment with keratanase. An even greater decrease in binding is observed after treatment with alkaline borohydride. A comparison of binding of antibody AN9P1 with that of another previously described monoclonal antibody, 1/20/5-D-4, to keratan sulphate [Caterson, Christner & Baker (1983) J. Biol. Chem. 258, 8848-8854] revealed similar binding characteristics, both showing much diminished binding after papain digestion of proteoglycan and even less with purified skeletal keratan sulphate. Removal of the Fc piece of antibody AN9P1 had no significant effect on the differential binding of divalent F(ab')2 fragment to proteoglycan, to papain-digested proteoglycan and to keratan sulphate, although there was a small decrease in binding to papain-digested proteoglycan. Conversion of the antibody into univalent Fab fragment with removal of the Fc piece resulted in diminished binding to proteoglycan, compared with that observed with IgG, and in enhanced binding to free keratan sulphate and to papain-digested proteoglycan. These results suggest that close proximity of keratan sulphate chains on the core protein of proteoglycans favours preferential reactivity of bivalent antibody with these species through cross-bridging of chains by antibody. Conversely, much decreased binding to keratan sulphate on proteoglycan core-protein fragments and to free keratan sulphate results from a lack of close proximity of keratan sulphate. By using univalent Fab fragment in these assays these differences in binding are minimized by preventing cross-bridging and thereby enhancing detection of smaller fragments without sacrificing too much sensitivity of detection of larger proteoglycan species. The persistent preferential binding of Fab fragment to proteoglycan is probably in part the result of the increased epitope density in the intact molecule compared with keratan sulphate in a more disperse form.     

Monoclonal antibodies specific for keratan sulfate detect epithelial-associated carbohydrates.

Monoclonal antibodies that specifically recognize epitopes on keratan sulfate glycosaminoglycans were used in this study to identify carbohydrate epitopes associated with many, but not all, types of epithelial cells. Immunoreactive cells included: keratinocytes, sebaceous gland cells, eccrine sweat gland duct cells, salivary gland excretory duct cells, colon adenocarcinoma cells, embryonic chick lung epithelial cells, embryonic chick mesonephric and metanephric kidney epithelial cells, and selected embryonic chick neural tube cells. Depending upon the type of epithelium, epitopes were located either within the cytoplasm or were located on cell surfaces. These epitopes were shared by cells from both human and chick tissues, indicating the absence of species specificity. Not all anti-keratan sulfate antibodies were equally effective in identifying epithelial-associated epitopes. One of the seven antibodies employed in this study failed to detect epitopes in almost all epithelial tissues studied. Of the remaining six antibodies, three were more effective than the others in recognizing epithelial-associated epitopes. These data indicate that carbohydrates that are typically associated with extracellular matrix can also be associated with epithelial cells, but in a form that is not necessarily related to extracellular matrix. These antibodies should prove to be useful in studies of the development of epithelial cells and tissues.     

A monoclonal antibody raised against Alzheimer cortex that specifically recognizes a subpopulation of microglial cells

A monoclonal antibody (MAb), termed AMC30, was raised after in vitro immunization with sonicated neurofibrillary tangle (NFT)-enriched fractions prepared from Alzheimer's brain. The antigen to which AMC30 is directed was expressed by microglial cells in senile plaques of Alzheimer's disease (AD). Microglia in the parenchyma surrounding brain tumors or infarctions, multinuclear giant cells, perivascular and parenchymal macrophages throughout the brain of AIDS patients were also labeled. Different non-nervous system lesions in which macrophages participate were also stained. Microglial cells in normal areas of the cortex or white matter were not labeled with MAb AMC30. The antigen to which AMC30 is directed was not detected in normal bone marrow, lymph nodes, lung, or spleen monocytes or macrophages. The epitope recognized by MAb AMC30 was present after formalin fixation and paraffin embedding. Our findings suggest that this MAb is directed against an antigen that is specifically expressed in a subpopulation of microglial cells and macrophages reactive to various pathological conditions.     

Production and characterization of monoclonal antibody to dermatan sulfate proteoglycan

We purified dermatan sulfate proteoglycan (PG) from the capsule of human ovarian fibroma for use as an immunogen. A monoclonal antibody, designated 6B6, was produced which reacts to the intact molecule of dermatan sulfate PG and the chondroitinase AC-treated core molecule on Western-blotted nitrocellulose membrane. Localization of materials showing crossreactivity to this antibody was studied in human tissues by indirect immunohistochemistry. The interstitial elements of almost all tissues examined were positive for the antibody: dermis, submucosal layer of digestive tract, perichondral layer, perivascular connective tissue, perineurium, adventitia of aorta, vessel wall of vein, pleura, and fibrous capsule of kidney and liver. Positive staining was also observed in fibrous elements at post-necrotic foci of cardiac muscle and pancreas, and at atherosclerotic lesions of aorta. The distribution of the antigen, core protein of the dermatan sulfate PG, revealed with 6B6 was compared to that of the dermatan sulfate side chain, which was demonstrated with antibody 9A-2 (Couchman et al.: Nature 307:650, 1984) after treatment with chondroitin sulfate B-lyase. The distribution of both antigens, core protein, and dermatan sulfate side chains showed the same pattern, with minor exceptions. The antibody 6B6 will be a useful tool to study the immunohistochemical localization of dermatan sulfate PG.     

Two subpopulations of differentiated chondrocytes identified with a monoclonal antibody to keratan sulfate

We have prepared a monoclonal antibody, named MZ15, that specifically binds keratan sulfate. Immunofluorescence studies showed that the distribution of keratan sulfate in articular cartilage was not uniform: the amount of keratan sulfate increased with distance from the articular surface. Two subpopulations of chondrocytes could be distinguished after isolation from cartilage by the presence or absence of cell surface keratan sulfate. Keratan sulfate-negative chondrocytes were shown to come from the upper cartilage layers. There was therefore a direct correlation between biochemical heterogeneity of cartilage matrix and heterogeneity within the chondrocyte population. During growth in monolayer culture, superficial chondrocytes began to synthesize keratan sulfate, but the cells could still be distinguished from cultures of deep or unfractionated chondrocytes by their reduced substrate adhesiveness and tendency to remain rounded.     

Antigenic properties of keratan sulfate: influence of antigen structure, monoclonal antibodies, and antibody valency.

The influence of (a) antigen structure, (b) type of monoclonal antibody, and (c) antibody bivalency on the immunochemical detection and quantification of keratan sulfate (KS) from aggrecan has been studied. Apparent KS epitope levels were determined by immunoglobulin G (IgG)-enzyme-linked immunosorbent assay (ELISA) in preparations of human aggrecan and in a defined series of lower molecular weight proteoglycan preparations generated by proteolytic and alkali treatment of aggrecan. Gel filtration chromatography showed KS epitope to be preferentially detected in the higher molecular weight fragments of the preparations. In single KS chains the epitope was detected in the chains of higher M(r). The ability of the proteoglycan to inhibit in the IgG-ELISA decreased with a reduction in proteoglycan fragment size, ranging between 6- and 260-fold, depending on the antibody used. This was considered to be a cooperative binding effect. With most antibodies, the sensitivity of the IgG-ELISA (represented by the steepness of the inhibition slope) was also reduced with smaller inhibitor sizes. The lowest limit of detectability (the amount of KS required to generate 20% inhibition) varied by up to 60-fold depending on the antibody used. The use of monovalent Fab fragments instead of the whole IgG anti-KS antibody in the ELISA showed that the bivalency of the antibody also affected the quantitation of the assay. In the Fab-ELISA the assay was found to have an increased detectability (by 9.5-fold with aggrecan as the inhibitor), and the proteoglycan fragments and aggrecan all generated parallel inhibition curves. Although the Fab-ELISA was somewhat influenced by the structural presentation of the KS, this was not apparent for small fragments and single chains. Thus the effects of cooperative binding and antibody valency could be overcome and quantitative data could be obtained for all samples, using papain-digested samples and the Fab-ELISA. Application of this assay to analysis of body fluids showed the KS-containing fragments in synovial fluid, serum, and urine were of different sizes and could be quantified.     

Arterial chondroitin sulfate proteoglycan: localization with a monoclonal antibody

We generated a monoclonal antibody (Mab) against a large chondroitin sulfate proteoglycan (CSPG) isolated from bovine aorta. This Mab (941) immunoprecipitates a CSPG synthesized by cultured monkey arterial smooth muscle cells. The immunoprecipitated CSPG is totally susceptible to chondroitinase ABC digestion and possesses a core glycoprotein of Mr approximately 400-500 KD. By use of immunofluorescence light microscopy and immunogold electron microscopy, the PG recognized by this Mab was shown to be deposited in the extracellular matrix of monkey arterial smooth muscle cell cultures in clusters which were not part of other fibrous matrix components and not associated with the cell's plasma membrane. With similar immunolocalization techniques, the CSPG antigen was found enriched in the intima and present in the medial portions of normal blood vessels, as well as in the interstitial matrix of thickened intimal lesions of atherosclerotic vessels. Immunoelectron microscopy revealed that this CSPG was confined principally to the space within the extracellular matrix not occupied by other matrix components, such as collagen and elastic fibers. These results indicate that this particular proteoglycan has a specific but restricted distribution in the extracellular matrix of arterial tissue.     

Altered fine structures of corneal and skeletal keratan sulfate and chondroitin/dermatan sulfate in macular corneal dystrophy

The content and fine structure of keratan and chondroitin/dermatan sulfate in normal human corneas and corneas affected by macular corneal dystrophies (MCD) types I and II were examined by fluorophore-assisted carbohydrate electrophoresis. Normal tissues (n = 11) contained 15 microg of keratan sulfate and 8 microg of chondroitin/dermatan sulfate per mg dry weight. Keratan sulfates consisted of approximately 4% unsulfated, 42% monosulfated, and 54% disulfated disaccharides with number of average chain lengths of approximately 14 disaccharides. Chondroitin/dermatan sulfates were significantly longer, approximately 40 disaccharides per chain, and consisted of approximately 64% unsulfated, 28% 4-sulfated, and 8% 6-sulfated disaccharides. The fine structural parameters were altered in all diseased tissues. Keratan sulfate chain size was reduced to 3-4 disaccharides; chain sulfation was absent in MCD type I corneas and cartilages, and sulfation of both GlcNAc and Gal was significantly reduced in MCD type II. Chondroitin/dermatan sulfate chain sizes were also decreased in all diseased corneas to approximately 15 disaccharides, and the contents of 4- and 6-sulfated disaccharides were proportionally increased. Tissue concentrations (nanomole of chains per mg dry weight) of all glycosaminoglycan types were affected in the disease types. Keratan sulfate chain concentrations were reduced by approximately 24 and approximately 75% in type I corneas and cartilages, respectively, and by approximately 50% in type II corneas. Conversely, chondroitin/dermatan sulfate chain concentrations were increased by 60-70% in types I and II corneas. Such changes imply a modified tissue content of individual proteoglycans and/or an altered efficiency of chain substitution on the core proteins. Together with the finding that hyaluronan, not normally present in healthy adult corneas, was also detected in both disease subtypes, the data support the conclusion that a wide range of keratocyte-specific proteoglycan and glycosaminoglycan remodeling processes are activated during degeneration of the stromal matrix in the macular corneal dystrophies.     

Production and characterization of monoclonal antibodies that specifically bind to phosphatidylcholine.

A series of monoclonal antibodies (mAbs) that react with phosphatidylcholine (PC) were established. All mAbs were highly specific to PC and no cross-reaction with other phospholipids were observed. The results obtained with two typical monoclonal antibodies, JE-1 and JE-8, were described. The analysis using synthetic PC analogs with modified polar head groups showed that the methyl groups on the quaternary nitrogen of the choline moiety were important for the binding. Each mAbs showed distinct acyl chain specificities of the PC molecules, and JE-1 showed considerable reactivity with PC with saturated fatty acids, whereas JE-8 could not react with the PC. Both mAbs bound to PC with unsaturated fatty acids, but showed distinct reactivity profiles. Both mAbs reacted only weakly with water-soluble haptens such as phosphorylcholine and L-alpha-glycerophosphocholine, suggesting that the hydrophobic moiety of the PC molecule is important for the maximum affinity. The interaction between the mAbs and the hydrophobic moieties of PC molecules was further studied by analyzing the effect of the mAbs on the activities of phospholipase A2 and phospholipase C. JE-1 inhibited both enzyme activities, while JE-8 inhibited only the phospholipase C activity, indicating that JE-1 interacts more thoroughly with the hydrophobic region of the PC molecule than JE-8 does.     

Monoclonal antibody that recognizes an epitope of the sperm equatorial region and specifically inhibits sperm-oolemma fusion but not binding.

Balb/c mice were immunized with purified hamster sperm heads for induction of antisera and the production of monoclonal antibodies that recognize preferentially the equatorial segment. Twenty-six hybridoma clones secreted monoclonal antibodies with strong affinity for spermatozoa. The supernatants of 16 clones contained antibodies against the equatorial segment, of which 11 were specific to this region. Five supernatants (M1-M5) containing antibodies that bind to various regions of the sperm head were selected and assessed for the ability to inhibit hamster fertilization in vitro using intact and zona-free oocytes. All the supernatants inhibited fertilization compared with the control. However, M1 supernatant specifically inhibited sperm-egg fusion in a concentration-dependent manner, while sperm-oolemma binding and sperm motility remained unaffected. M1 supernatant recognized an epitope that is exclusive to the equatorial segment and expression of this epitope increased after capacitation and the acrosome reaction. Preliminary immunoblot analysis indicated that M1 monoclonal antibody recognized two protein bands of 37.5 and 34.0 kDa.     

Analysis of human C8 with monoclonal antibodies. Characterization of a monoclonal antibody that recognizes free C8 alpha-gamma subunit

The eighth component of human C is essential for the formation of the membranolytic C attack complex. C8 has a unique structure in that two covalently linked chains, C8 alpha and C8 gamma, are associated non-covalently with the third chain, C8 beta. In order to study the structure and assembly of the C8 molecule, a panel of mAb has been produced against the C component C8. Eight of these mAb had reactivity to the C8 alpha-gamma subunit, whereas four reacted with C8 beta. One of the C8 alpha-gamma mAb, C8A2, had specificity for an epitope on the C8 alpha-chain and exhibited no cross-reactivity to any of the other terminal C components, including C8 beta. C8A2 inhibited the hemolytic activity of the C8 alpha-gamma subunit but had no effect on the activity of fluid phase whole C8 or C8 within membrane-bound C5b-8. Functional experiments suggest that C8A2 inhibits C8 alpha-gamma activity by interfering with its interaction with the C8 beta-chain. In an enzyme immunoassay using the C8A2 mAb, free C8 alpha-gamma subunit could be detected in both homozygous and heterozygous C8 beta-deficient serum. However, only low level binding was observed when homozygous C5- and C7-deficient sera were tested. Thus the mAb, C8A2, recognizes an epitope expressed on the C8 alpha-gamma subunit but not on intact C8 and can detect free C8 alpha-gamma in the presence of native C8.