Immunogenicity of xenogeneic cartilage matrix components in a rabbit model

Purified xenogeneic cartilage matrix components, including proteoglycan subunits, chondroitin 4 sulfate, and chondroitin 6 sulfate, were inoculated into the knee joint of rabbits, and local as well as systemic responses were evaluated. proteoglycan was associated with synovial hyperplasia and infiltrates of eosinophils and lymphocytes and with rising titers of antiproteoglycan antibodies in a tanned sheep rbc hemagglutination assay over a six-week period of weekly intra-articular injections and observations. Chondroitin sulfates failed to evoke detectable changes in the joint or serum. Immunogenicity of cartilage matrix components may play a role in allogeneic and xenogeneic osteochondral grafts, and it is also possible that autogenous matrix immunogens exist and contribute to progression of degenerative joint disease. The immunogenicity of allogeneic and autogenous cartilage matrix components remains undefined.     

紫藤凝集素的分离纯化及理化性质研究

用常规方法处理的ＤＥＡＥ离子交换纤维素柱,通过线性离子强度梯度洗脱,从紫藤种子的蛋白粗提液中得到一定纯度的紫藤凝集素。纯化的凝集素凝集兔红血球的比活提高４０倍,总活力回收率为１９．２％。紫藤凝集素的分子量经ＰＡＧＥ鉴定为２０５ｋｄ,是由两种亚基构成的四聚体,这两种亚基各有２个,分子量ＳＤＳ－ＰＡＧＥ鉴定分别为７７６００ｄ和２５１００ｄ。紫藤凝集素是一种糖蛋白,等电点约为４．６０。它可凝集人的各种血     

Vitronectin and thrombospondin promote retinal neurite outgrowth: developmental regulation and role of integrins.

Extracellular matrix (ECM) glycoproteins regulate neuronal development and axonal growth. In this paper, the ECM glycoprotein vitronectin was identified and localized in the embryonic chick neuroretina. To identify potentially important neurite outgrowth-promoting molecules, responses of embryonic chick retinal neurons to vitronectin and thrombospondin, another retinal ECM constituent, were examined. These neurons were shown to attach and extend neurites on either glycoprotein. Integrins containing the alpha v or beta 1 subunits mediate both responses to vitronectin and neurite outgrowth on thrombospondin. Attachment to thrombospondin was inhibited by heparin, suggesting that neurons also utilize a proteoglycan or sulfated glycolipid as a receptor for this glycoprotein. Thus, retinal neurons use specific receptors to interact with vitronectin and thrombospondin, two glycoproteins present in the embryonic neuroretina, suggesting roles for these ligands and their receptors in retinal development.     

Purification of the most abundant protein in the coelomic fluid of a sea urchin which immunologically cross reacts with 23S glycoprotein in the sea urchin eggs

The most abundant glycoprotein in the coelomic fluid of sea urchin Anthocidaris crassispina was purified and its subunit structure, molecular form in the native state, amino acid composition, and electron micrographic image were studied. The results showed that the protein in its native state was basically a tetramer with a total molecular weight of about 700,000, which was in equilibrium with a high molecular weight form corresponding to an octamer. The electron micrograph of the tetramer showed two ellipsoidal units aligned in parallel with a wide gap in between. The subunits all had the same molecular weight of 180,000 +/- 10,000 and were disulfide bonded in pairs. The carbohydrate content was about 16% with mannose and fucose as the two most abundant sugars. Although this protein accounted for 70% of the total protein in the coelomic fluid, it did not take part in the known activities of the fluid, namely hemagglutination and coagulation. Despite its structural similarity to the mammalian alpha-2-macroglobulin or reptilian and avian ovomacroglobulins it did not interact with bovine trypsin or chymotrypsin. This protein showed immunological cross reactivity with 23S glycoprotein purified from sea urchin eggs which, we believe, corresponds to the previously described 22-27S protein particles in eggs.     

Electron microscopic studies of proteoglycan aggregates from bovine articular cartilage.

Proteoglycan aggregates from bovine articular cartilage have been visualized by electron microscopy of mixed proteoglycan-cytochrome c monolayers. The proteoglycan aggregates consist of proteoglycan subunits arising laterally at fairly regular intervals (20 to 30 nm) from the opposite sides of an elongated filamentous structure. The filamentous backbone in individual aggregates varies in length from 400 to 4000 nm. The individual proteoglycan subunits in the aggregate vary in length from 100 to 400 nm. However, there is no difference in the average size of the proteoglycan subunits associated with the largest or smallest aggregates. The sizes of the individual aggregates are determined mainly by the lengths of their filamentous backbones. The stoichiometry of binding of subunits to filament, calculated from the data reported here, is close to that for the binding of subunits to hyaluronic acid reported by others.     

Purification and characterization of a lectin (plant hemagglutinin) with N blood group specificity from Vicia graminea seeds.

A lectin with N blood group specificity was isolated from Vicia graminea seeds. This lectin was purified from a crude extract by precipitation with ammonium sulfate, DEAE-cellulose chromatography and Sephadex G-150 gel filtration. Purification steps were followed by increase of specific activity. Its homogeneity was demonstrated by polyacrylamide gel electrophoresis, immunoelectrophoresis, electrofocusing and ultracentrifugation. This lectin is an acid glycoprotein with 7.3% carbohydrate, a high percentage of serine and contains no sialic acid. The native lectin has a molecular weight about 100 000 and dissociates into four subunits of 25 000 as shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Preliminary hemagglutination inhibition has shown that the lectin was not inhibited by any of the monosaccharides contained in N blood group substances; however it was inhibited by the erythrocyte membrane major glycoprotein and the tryptic fragments obtained from erythrocytes.     

Molecular size distribution of proteoglycan subunits and glycosaminoglycans synthesized by chondrocytes under conditions of reduced proteoglycan synthesis

The rate of proteoglycan synthesis by chondrocytes in vitro was depressed by either omitting l-glutamine from the incubation medium or by addition of proteoglycan subunit to the medium. The molecular size distribution on Sepharose 2B of the proteoglycan subunits synthesized by the chondrocytes under these conditions of reduced proteoglycan synthesis was found to be the same as those synthesized by the control cells. Likewise, the molecular size distribution on Sepharose 6B CL of the glycosaminoglycan chains synthesized by the depressed cells was found to be similar to that observed in untreated chondrocytes. This work demonstrates that, under conditions of reduced proteoglycan synthesis, fewer proteoglycan subunits are synthesized by chondrocytes and that the molecular size distribution of these macromolecules is similar to those synthesized by untreated cells.     

The predominant form of secreted colony stimulating factor-1 is a proteoglycan.

Colony stimulating factor-1 (CSF-1) is a homodimeric glycoprotein that humorally regulates the proliferation and differentiation of mononuclear phagocytic cells and locally regulates cells of the female reproductive tract. Alternative splicing of the human CSF-1 mRNA leads to alternative expression of the CSF-1 homodimer as a secreted glycoprotein or as a membrane-spanning molecule with cell surface biological activity. In the present study, analysis of immunoaffinity-purified CSF-1 from mouse L929 cell medium by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that CSF-1 is predominantly secreted as highly sulfated species of 375- and 250-kDa with a smaller amount of a 100-kDa species. Analysis by gel filtration in 4 M guanidine HCI buffer, indicated that, in contrast to the 100-kDa species, the highly sulfated species exhibit anomalously high molecular weights and self-association on SDS-PAGE similar to the dermatan sulfate proteoglycan, biglycan. The three predominant CSF-1 species were shown to be an 80-kDa homodimer, an 80-kDa/50-kDa heterodimer, and a 50-kDa homodimer. The 80-kDa subunit contained a single 18-kDa chondroitin sulfate chain that was absent from the 50-kDa subunit. Furthermore, treatment of the 80- and 50-kDa subunits, synthesized in the presence of tunicamycin, with chondroitinase ABC, neuraminidase, and endo-alpha-N-acetyl galactosaminidase reduced their apparent molecular masses to 60 and 25 kDa, respectively. These results are consistent with intracellular proteolytic cleavage of the 80-kDa chondroitin sulfate containing subunits from the membrane spanning CSF-1 precursor at a point carboxyl-terminal to the single consensus sequence for glycosaminoglycan addition and cleavage of the 50-kDa glycoprotein subunit at a position aminoterminal to this site. The predominance of the proteoglycan form of secreted CSF-1, which represents only 3-4% of the total trichloroacetic acid-precipitable counts released from 35SO4(2-)-labeled L cells, has important implications for regulation by this growth factor.     

Isolation and characterisation of a sialic acid-binding lectin (carcinoscorpin) from Indian horseshoe crab Carcinoscorpius rotunda cauda

A sialic acid-binding lectin, carcinoscorpin, has been purified to apparent homogeneity in 40% yield from the Indian horseshoe carb, Carcinoscorpius rotunda cauda. This glycoprotein lectin of molecular weight 420,000 was composed of two non-identical subunits of molecular weights 27,000 and 28,000 as determined by gel electrophoresis in the presence of sodium dodecyl sulfate. The hemagglutination activity of the lectin was susceptible to guanidine-HCl; modification of tyrosyl and tryptophanyl residues also inhibited the activity although alkylation of the -SH group, reduction of disulfide bonds or modification of amino and carboxyl groups were without any effect. The monomeric form of the lectin produced by succinylation of native protein was inactive in binding to sialoglycoconjugates.     

Distinct in vivo roles of colony-stimulating factor-1 isoforms in renal inflammation

CSF-1, the major regulator of macrophage (Mphi) development, has three biologically active isoforms: a membrane-spanning, cell surface glycoprotein, a secreted glycoprotein, and a secreted proteoglycan. We hypothesized that there are shared and unique roles of individual CSF-1 isoforms during renal inflammation. To test this, we evaluated transgenic mice only expressing the cell surface or precursors of the secreted CSF-1 isoforms for Mphi accumulation, activation, and Mphi-mediated tubular epithelial cell (TEC) apoptosis during unilateral ureteral obstruction. The only difference between secreted proteoglycan and secreted glycoprotein CSF-1 isoforms is the presence (proteoglycan) or absence (glycoprotein) of an 18-kDa chondroitin sulfate glycosaminoglycan. We report that 1) cell surface CSF-1 isoform is sufficient to restore Mphi accumulation, activation, and TEC apoptosis to wild-type levels and is substantially more effective than the secreted CSF-1 isoforms; 2) the chondroitin sulfate glycosaminoglycan facilitates Mphi accumulation, activation, and TEC apoptosis; 3) increasing the level of secreted proteoglycan CSF-1 in serum amplifies renal inflammation; and 4) cell-cell contact is required for Mphi to up-regulate CSF-1-dependent expression of IFN-gamma. Taken together, we have identified central roles for the cell surface CSF-1 and the chondroitin sulfate chain on secreted proteoglycan CSF-1 during renal inflammation.     

The role of link protein in mediating the interaction between hyaluronic acid and newly secreted proteoglycan subunits from adult human articular cartilage

Normal adult human articular cartilage in organ culture secretes proteoglycan subunits that cannot initially interact in a normal manner with hyaluronic acid unless the latter is present at high concentrations and a neutral pH is employed. However, if the newly secreted subunit is allowed to mature in the cartilage matrix for up to 12 h, then its ability to interact is indistinguishable from that of its more mature counterparts. This conversion does not take place if the proteoglycan subunits are incubated in dilute solutions in the absence of the cartilage, and it is prevented by culturing at low temperature. The newly secreted proteoglycan subunits can, however, be induced to interact with hyaluronic acid by the presence of link proteins. The complex formed by these three components cannot be dissociated in the presence of hyaluronic acid oligosaccharides, suggesting a normal aggregate configuration. It is thus possible that proteoglycan aggregate formation within the cartilage is initially mediated by the presence of link proteins, which induce a conformational change with the hyaluronic acid-binding region of the proteoglycan subunits, although additional modification may be necessary to render any such change irreversible.     

Structural analysis of glycopeptides by polyacrylamide gel electrophoresis

Three different well-characterized preparations of proteoglycan subunits were analyzed by high-performance liquid chromatography on a silica-based material bonded with an amide phase. The biochemical integrity of the proteoglycan subunits was retained during this procedure. The high sensitivity coupled with the increased speed of high-performance liquid chromatography will permit rapid analysis and comparisons of very small specimens.     

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.     

Multiple aggregation factors in cartilage proteoglycan (Short Communication)

Proteoglycan as isolated from bovine nasal septa is a partially aggregated complex containing linking factors. Two essential factors can be separated from proteoglycan subunits and resolved in a CsCl density gradient. Chondroitinase digestion of the purified proteoglycan subunits does not interfere with their aggregation when the linking factors are added.     

Isolation of a cell-surface receptor for chick neural retina adherons

Embryonic chick neural retina cells release glycoprotein complexes, termed adherons, into their culture medium. When absorbed onto the surface of petri dishes, neural retina adherons increase the initial rate of neural retina cell adhesion. In solution they increase the rate of cell-cell aggregation. Cell-cell and adheron-cell adhesions of cultured retina cells are selectively inhibited by heparan-sulfate glycosaminoglycan, but not by chondroitin sulfate or hyaluronic acid, suggesting that a heparan-sulfate proteoglycan may be involved in the adhesion process. We isolated a heparan-sulfate proteoglycan from the growth-conditioned medium of neural retina cells, and prepared an antiserum against it. Monovalent Fab' fragments of these antibodies completely inhibited cell-adheron adhesion, and partially blocked spontaneous cell-cell aggregation. An antigenically and structurally similar heparan-sulfate proteoglycan was isolated from the cell surface. This proteoglycan bound directly to adherons, and when absorbed to plastic, stimulated cell-substratum adhesion. These data suggest that a heparan-sulfate proteoglycan on the surface of chick neural retina cells acted as a receptor for adhesion-mediating glycoprotein complexes (adherons).     

Serum i antigen: A new human blood-group glycoprotein

A glycoprotein has been found in normal serum which inhibits the hemagglutination caused by human anti-i cold agglutinins. This glycoprotein binds to a Sepharose anti-i affinity column at 4°C and can be quantitatively eluted at 37°C. The eluted glycoprotein is specific in that it has potent hemagglutination-inhibition activity against anti-i antibodies but does not inhibit the hemagglutination by anti-I, -A, -B, -H or by the lectins Con A, PHA or Vicia graminea. The anti-i affinity method should provide a simple procedure for large scale purification and subsequent characterization of the i-antigen.     

Studies on the interaction of newly secreted proteoglycan subunits with hyaluronate in human articular cartilage

Newly secreted proteoglycans from adult human cartilage do not interact well with hyaluronate, but attain this ability with time in the extracellular matrix. The conversion process occurs in all types of cartilagenous matrix, as newborn cartilage cultures, chondrosarcoma cultures and adult chondrocyte cultures each secreted proteoglycan subunits which exhibited the delayed aggregation phenomenon. However, the rate of conversion is probably dependent upon the structure of the surrounding matrix and the cell type. In vitro, link protein appears to enhance an initial change in the hyaluronate-binding region of the newly secreted proteoglycan subunits to allows stronger interaction with hyaluronate. In a second step, which is pH- and temperature-dependent, the change becomes irreversible. Thus, in addition to its role in stabilizing the interaction of mature proteoglycan subunits with hyaluronate, link protein may also aid in promoting the conversion of the newly synthesized proteoglycan subunit to a form that is capable of strong interaction with hyaluronate.     

A lectin from the lichenized Basidiomycete Dictyonema glabratum

A lectin with hemagglutinating activity has been isolated from an aqueous extract of the symbiotic phenotype of Dictyonema glabratum and its cyanobacterial photobiont Scytonema sp. The purified lectin had a pI of 6.8 and its molecular mass was investigated by electrospray ionization mass spectrometry, gel filtration and SDS-PAGE, which indicated its native conformation as a dimer formed by two identical subunits of 16540 Da. The lectin is a glycoprotein with a low degree of glycosylation, containing galactose, xylose, glucose and mannose as neutral monosaccharides, in addition to glucosamine, which could indicate both N - and O -linkages. Amino acid analysis showed the predominance of nonpolar residues such as phenylalanine. Agglutination of human erythrocytes required divalent cations, which is affected by addition of EDTA. The lectin was more stable at 30 °C or less for at least 1 h and between pH 5.0 and 7.0. Among the various compounds tested for hemagglutination inhibition, N -acetylgalactosamine was the most active. The potential role of this lectin in recognition of the compatible cyanobacterial photobiont is discussed.     

Isolation and characterization of developmentally regulated chondroitin sulfate and chondroitin/keratan sulfate proteoglycans of brain identified with monoclonal antibodies

A panel of monoclonal antibodies prepared to the chondroitin sulfate proteoglycans of rat brain was used for their immunocytochemical localization and isolation of individual proteoglycan species by immunoaffinity chromatography. One of these proteoglycans (designated 1D1) consists of a major component with an average molecular size of 300 kDa in 7-day brain, containing a 245-kDa core glycoprotein and an average of three 22-kDa chondroitin sulfate chains. A 1D1 proteoglycan of approximately 180 kDa with a 150-kDa core glycoprotein is also present at 7 days, and by 2-3 weeks postnatal this becomes the major species, containing a single 32-kDa chondroitin 4-sulfate chain. The concentration of 1D1 decreases during development, from 20% of the total chondroitin sulfate proteoglycan protein (0.1 mg/g brain) at 7 days postnatal to 6% in adult brain. A 45-kDa protein which is recognized by the 8A4 monoclonal antibody to rat chondrosarcoma link protein copurifies with the 1D1 proteoglycan, which aggregates to a significant extent with hyaluronic acid. A chondroitin/keratan sulfate proteoglycan (designated 3H1) with a size of approximately 500 kDa was isolated from rat brain using monoclonal antibodies to the keratan sulfate chains. The core glycoprotein obtained after treatment of the 3H1 proteoglycan with chondroitinase ABC and endo-beta-galactosidase decreases in size from approximately 360 kDa at 7 days to approximately 280 kDa in adult brain. In 7-day brain, the proteoglycan contains three to five 25-kDa chondroitin 4-sulfate chains and three to six 8.4-kDa keratan sulfate chains, whereas the adult brain proteoglycan contains two to four chondroitin 4-sulfate chains and eight to nine keratan sulfate chains, with an average size of 10 kDa. The concentration of 3H1 increases during development from 3% of the total soluble proteoglycan protein at 7 days to 11% in adult brain, and there is a developmental decrease in the branching and/or sulfation of the keratan sulfate chains. A third monoclonal antibody (3F8) was used to isolate a approximately 500-kDa chondroitin sulfate proteoglycan comprising a 400-kDa core glycoprotein and an average of four 28-kDa chondroitin sulfate chains. In the 1D1 and 3F8 proteoglycans of 7-day brain, 20 and 33%, respectively, of the chondroitin sulfate is 6-sulfated, whereas chondroitin 4-sulfate accounts for greater than 96% of the glycosaminoglycan chains in the adult brain proteoglycans.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification of a hyaluronic acid-binding protein that interferes with the preparation of high-buoyant-density proteoglycan aggregates from adult human articular cartilage.

Adult human articular cartilage contains a hyaluronic acid-binding protein of Mr 60 000-75 000, which contains disulphide bonds essential for this interaction. The molecule can compete with proteoglycan subunits for binding sites on hyaluronic acid, and can also displace proteoglycan subunits from hyaluronic acid if their interaction is not stabilized by the presence of link proteins. The abundance of this protein in the adult accounts for the reported inability to prepare high-buoyant-density proteoglycan aggregates from extracts of adult human cartilage [Roughley, White, Poole & Mort (1984) Biochem. J. 221, 637-644], whereas the deficiency of the protein in newborn human cartilage allows the normal recovery of proteoglycan aggregates from this tissue. The protein shares many common features with a hyaluronic acid-binding region derived by proteolytic treatment of a proteoglycan aggregate preparation, and this may also represent its origin in the cartilage, with its production increasing during tissue maturation.