An 18-kDa glycoprotein from bovine nasal cartilage. Isolation and primary structure of small, cartilage-derived glycoprotein

A glycosylated protein (small, cartilage-derived glycoprotein, SCGP) of approximately 18 kDa with unknown function has been isolated from dissociative extracts of bovine nasal cartilage and its primary structure determined. The protein has 121 amino acids, giving a calculated protein molecular weight of 13,878, four disulfide bonds, two N-linked oligosaccharides and one O-linked oligosaccharide. In nasal cartilage, this glycoprotein is in molar concentrations equivalent to 1/5-1/2 that of the link protein of cartilage proteoglycan aggregates, and it has also been isolated from bovine articular cartilage and from bovine fetal epiphysis. The N-terminal, glycosylated region of the molecule is relatively rich in arginine, proline, glycine, and threonine. The C-terminal 82 amino acids (which contains all four of the disulfide bonds and none of the carbohydrate) can be found as a discrete entity in cartilage extracts, indicating that the N-terminal domain is readily removed by extracellular proteolytic attack.     

Partial characterization of protocollagen from embryonic cartilage

1. Attempts were made to isolate and characterize the protocollagen that accumulates in connective tissue when the hydroxylation of proline and lysine is inhibited. The term protocollagen has been used to describe the proline-rich and lysine-rich polypeptide or polypeptides that serve as substrates for the formation of hydroxyproline and hydroxylysine during the synthesis of collagen. 2. Both protocollagen and newly synthesized collagen from embryonic cartilage were isolated as complex aggregates, which contained sulphated mucopolysaccharides and other proteins or polypeptides from the same tissue. The complexes containing protocollagen were similar to those containing newly synthesized collagen when examined with several different techniques. 3. After the complexes were denatured and disaggregated, zone centrifugation and gel filtration indicated that the denatured protocollagen was similar to the denatured newly synthesized collagen obtained from cartilage in which the hydroxylation was not inhibited, and it was also similar to purified alpha-collagen. The results suggest that, when the hydroxylation is inhibited, most of the protocollagen polypeptides that accumulate are as large as complete alpha-chains of collagen. 4. Significant purification of the protocollagen polypeptides was obtained with a new technique for DEAE-Sephadex chromatography in which urea was used to prevent aggregation of the samples and the column was eluted with guanidine thiocyanate. 5. Protocollagen polypeptides were completely hydrolysed to diffusible peptides by a specific collagenase. 6. It is not entirely clear whether the hydroxylation normally begins while relatively short protocollagen molecules are still attached to polysomes, or whether protocollagen molecules of the size of alpha-collagen are synthesized even when the hydroxylation is not inhibited. 7. Results obtained with puromycin suggest that some hydroxylation occurs with smaller polypeptides, but polypeptide chains approaching the size of alpha-collagen are required to obtain complete hydroxylation of the appropriate amino acid residues of protocollagen.     

Properties of highly purified lysyl oxidase from embryonic chick cartilage.

Lysyl oxidase the enzyme which oxidately deaminates lysine residues in collagen and elastin, was purified from embryonic chick cartialge by employing an affinity column of lathyritic rat skin collagen coupled to Sepharose, followed by separation on DEAE-cellulose. An enzyme preparation was obtained which was pure as shown by polyacrylamide gel electrophoresis. The specific activity was 1800-fold higher than that of the original extract. The pure enzyme utilized both collagen and elastin substrate. Furthermore, the ratios of enzyme activity with elastin substrate versus that with collagen substrate were the same at all stages of purity. Only one protein band was found after polyacrylamide gel electrophoresis of the pure lysyl oxidase in sodium dodecyl sulfate and mercaptoethanol. The molecular weight was estimated to be 28000. It was found that the enzyme contained a large number of cysteine and tyrosine residues. Evidence was obtained for molecular heterogeneity of lysyl oxidase. The enzyme eluted from DEAE-cellulsoe in at least four distinct regions. When the peaks were rechromatographed separately, they eluted at salt concentrations similar to those of the original chromatogram. However, the substrate specificity and the electrophoretic mobility on polyacrylamide gel were the same for all enzyme fractions.     

A major Echinococcus multilocularis antigen is a mucin-type glycoprotein.

The metacestode of Echinococcus multilocularis is surrounded by a carbohydrate-rich laminated layer, which plays a key role in the establishment of the infection in the mammalian host. A major component of the laminated layer is an antigen referred to as Em2(G11). This highly species-specific antigen has been used for serodiagnoses of alveolar echinococcosis and is suggested to contain carbohydrates as major constituents. The results of this work have shown that immunoaffinity-purified Em2(G11) subjected to size-exclusion chromatography eluted mainly in the void volume, indicating a high molecular weight structure of this antigen. Amino acid analysis revealed a large proportion of threonine and proline residues in Em2(G11). The carbohydrate moiety of the antigen was found to be composed of galactose, N-acetylgalactosamine, and N-acetylglucosamine with a ratio of 2.4:1.0:0.5 as determined by gas-chromatography/mass spectrometry. An isotope tag was introduced to the beta-eliminated glycans, and an integrated mass spectrometric O-glycan profiling and sequencing approach was employed to obtain detailed sequence and linkage information of the unseparated glycoform pool. Novel glycoforms containing mucin-type core Gal1-3GalNAc and branched core structures attached to both serine and threonine residues are described. The data presented reveal that the Em2(G11) antigen is a mucin-type glycosylated protein.     

Cytochalasin B releases a major surface-associated glycoprotein, fibronectin, from cultured fibroblasts

BHK21 cells cultured in minimal essential medium (Eagle) supplemented with 10% dialyzed fetal calf serum did not grow as they did in whole serum containing medium. Logarithmic growth was, however, initiated after a lag period, the length of which was dependent upon the cell density: medium volume ratio. The quiescent cells conditioned the medium during this lag period, and growth stimulation was apparently due to the release of serine into the medium. Cells cultured in 10% dialyzed serum plus the low molecular weight fraction of serum (serum dialysate), grew with kinetics similar to cells cultured in serum containing medium. When serum dialysate was chromatographed on Bio-gel P-2 the growth promoting activity eluted with the amino acids. Each of the non-essential amino acids was tested for its ability to stimulate the growth of cells in 10% dialyzed serum. Serine was capable of stimulating cell growth to the same extent as 10% serum dialysate and its concentration optimum was similar to its concentration in 10% serum dialysate. The remaining non-essential amino acids were either slightly stimulatory or had no effect on cell growth. Shifting a logarithmically growing population of cells to serine-free medium resulted in the accumulation of 95% of the cells in the G1 phase of the cell cycle within 24 h. Escape from the G1 block could occur if serine was added to the medium or if the cells were allowed to condition the medium. Entry of cells into S phase after the addition of 0.05 μmoles/ml of serine followed a 4–6 h lag and 80% of the cells were synthesizing DNA 12 h after shift-up.     

A cartilage catabolic factor from synovium.

Porcine synovium in organ culture produces a factor that causes chondrocytes to degrade their matrix. A quantitative assay for the factor, for which the cartilage of bovine nasal septum is used, is described. Evidence is presented that the catabolic factor is a protein.     

Temporal and spatial transitions in collagen types during embryonic chick limb development : II. Comparison of the embryonic cartilage collagen molecule with that from adult cartilage

Analyses were performed to compare the (α1)₃ collagen molecule synthesized by the cartilaginous long bone anlage of 8-day chick embryos with that produced by adult cartilage, [α1(II)]₃. The embryonic molecule produced segment-long-spacing crystallites which had a banding pattern indistinguishable from that of authentic [α1(II)]₃. After carboxymethyl cellulose chromatography, the material in the α1 chain peak of the embryonic collagen was found to have the same amino acid composition as that of the α1(II) chain.     

Sulfate incorporation from ascorbate 2-sulfate into chondroitin sulfate by embryonic chick cartilage epiphyses.

Radioactivity was significantly incorporated from ascorbate 2-[35S]sulfate into chondroitin sulfate by embryonic chick cartilage epiphyses. The extent of incorporation was comparable with that from inorganic [35S]sulfate. The radioactive chondroitin sulfate formed from ascorbate 2-[35S]sulfate gave two radioactive disaccharides on chondroitinase-ABC [EC 4.2.2.4] digestion. The incorporation was markedly decreased by inorganic sulfate. The time course of incorporation from ascorbate 2-[35S]sulfate and inorganic [35S]sulfate into chondroitin sulfate and the constituent disaccharides suggest that the incorporation rates from the two radioactive substances are different.     

Association and release of the major intrinsic membrane glycoprotein from peripheral nerve myelin.

Hypo-osmotic homogenization of the endoneurium from the adult-rat sciatic nerve and subsequent evaluation of the 197 000 g aqueous supernatant by sodium dodecyl sulphate pore-gradient electrophoresis (SDS-p.g.e.) revealed a release of the major glycoprotein (P0) (29 000 Mr) from peripheral nerve myelin. Immunological verification of the presence of this asparagine-linked glycoprotein in the aqueous supernatant was obtained by immune overlay after SDS-p.g.e. and electrophoretic transfer to nitrocellulose using anti-P0 gamma-globulin followed by autoradiographic detection with 125I-protein A. A comparison of successive hypo- and iso-osmotic extractions of the endoneurium revealed that the hypo-osmotic extraction released increasing amounts of P0 into the supernatant fraction, whereas the iso-osmotic treatment revealed lower levels of P0 extracted from the myelin and lesser amounts with each successive extraction. Three successive hypo-osmotic extractions resulted in a 2.0-, 2.9-, and 9.5-fold increase in the amount of P0 released compared with the successive iso-osmotic extractions. Although these results suggest that this major myelin glycoprotein has properties similar to those of extrinsic membrane proteins, temperature-dependent phase-partitioning experiments with Triton X-114 revealed that this glycoprotein is recovered in the detergent-enriched lower phase. These results indicate that this major myelin glycoprotein is an amphipathic integral membrane protein with a distinct hydrophobic domain and yet has solubility characteristics typical of an extrinsic membrane protein. P0 labelled in vitro with [3H]mannose could be immunoprecipitated from the aqueous supernatant with anti-P0 gamma-globulin by centrifugation at 197000g without the addition of second antibody or protein A. Analysis of such an immune precipitate after incorporation in vitro with [14C]acetate to label endoneurial lipids revealed that all major endoneurial lipid classes contained radioactive label, as determined by fluorography after high-performance t.l.c. The mechanism of release of this intrinsic glycoprotein from the myelin membrane, therefore, involves the osmotic-dependent formation of mixed micelles or membrane vesicles with endogenous membrane lipids.     

A human herpesvirus 7 glycoprotein, U21, diverts major histocompatibility complex class I molecules to lysosomes.

All members of the herpesvirus family persist in their host throughout life. In doing so, herpesviruses exploit a surprising number of different strategies to evade the immune system. Human herpesvirus 7 (HHV-7) is a relatively recently discovered member of the herpesvirus family, and little is known about how it escapes immune detection. Here we show that HHV-7 infection results in premature degradation of major histocompatibility complex class I molecules. We identify and characterize a protein from HHV-7, U21, that binds to and diverts properly folded class I molecules to a lysosomal compartment. Thus, U21 is likely to function in the normal course of HHV-7 infection to downregulate surface class I molecules and prevent recognition of infected cells by cytotoxic T lymphocytes.     

A fibronectin-binding glycoprotein from human platelet membranes.

Fibronectin ('cold-insoluble globulin') has been suggested as a possible mediator of platelet adhesion. A fibronectin-binding protein as partially purified from washed solubilized human platelet membranes by affinity chromatography on fibronectin-Sepharose. The isolated protein migrated as a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with an Mr (relative molecular mass) of approx. 125 000 under reducing conditions. The protein migrated as a dimer in non-reduced gels. The purified protein did not react with immunoglobulins against fibrinogen or fibronectin when tested in crossed immunoelectrophoresis or electroimmunoassay. The protein and purified fibronectin formed a complex that had a significantly faster mobility in crossed immunoelectrophoresis than did native fibronectin. The presence of heparin in the binding-protein-fibronectin mixture resulted in an even faster mobility of the complex, whereas the mobility of native fibronectin was unaffected. Crossed affinoimmunoelectrophoresis of the complex using different lectins suggested that the binding protein is a glycoprotein containing N-acetylglucosamine residues. The complex, but not purified fibronectin, bound to phenyl-Sepharose on crossed hydrophobic-interaction immunoelectrophoresis. The results strongly suggest the presence of a fibronectin-binding glycoprotein in the platelet membrane.     

Isolation of the major glycoprotein from fat cell plasma membranes

The major glycoprotein of rabbit fat cell plasma membranes has been solubilized by Brij 99 extraction and purified to homogeneity by preparative polyacrylamide gel electrophoresis and concanavalin A-Sepharose affinity chromatography. The isolation procedure yielded a glycoprotein with an apparent molecular weight of 79,000 which appeared as a single component by Coomassie blue and periodic acid-Schiff staining as well as by distribution of radioactivity after ¹²⁵I labeling. The lectin chromatography was effective in removing polypeptides and Schiff-nonreactive glycoproteins which migrated in close proximity to the major glycoprotein during electrophoresis but were not retained on the concanavalin A column. Determination of the amino acid and sugar composition of the purified glycoprotein indicated that it contained 18% carbohydrate by weight which occurred in the form of 30 mannose, 14 galactose, 23 glucosamine, 3 galactosamine, 6 N-acetylneuraminic acid, and 1 fucose residues per molecule. Approximately one-fifth of the total protein-bound saccharide of the adipocyte plasma membrane was accounted for by this glycoprotein and its composition suggested that it was the source of some of the previously identified (Y. Kawai, and R. G. Spiro, 1977, J. Biol. Chem.252, 6236–6244) asparagine- and serine (threonine)-linked carbohydrate units of the fat cell surface.     

The synthesis, turnover, and artificial restoration of a major cell surface glycoprotein.

A large glycoprotein (molecular weight equals 220,000 daltons) can be isolated from the surfaces of chick embryo fibroblasts. Such a protein had been shown by others to be present on normal cells in vitro, but missing from transformed variants of these cells, suggesting a possible role in growth control. We have estimated the turnover rate of this cell surface protein (CSP) and show that it is not more rapid than that of other cell proteins. We have also examined the resynthesis of CSP after removal from cells by trypsinization. This restoration is gradual, requiring more than 24 hr, and is affected by cell density. Restoration can be blocked by cycloheximide. Using cycloheximide to maintain cells depleted of CSP, we have demonstrated that isolated CSP can be progressively reabsorbed at 37 degrees C onto these denuded cell surfaces, and that this adsorption is not observed at 4 degrees C. This information on the turnover, depletion, and restoration of CSP may provide a means of directly testing its function.