Spinal fusion with recombinant human growth and differentiation factor-5 combined with a mineralized collagen matrix.

The availability of recombinant osteoinductive growth factors and new osteoconductive matrices offers an alternative to the use of autogenous bone (autograft) for grafting indications. This study evaluates the bone-forming activity of a mineralized collagen matrix combined with recombinant human growth and differentiation factor-5 in a rabbit posterolateral spinal fusion model. The activity of three distinct matrix-growth factor formulations is assessed by radiographic, histologic, and mechanical strength methods. Results show that the radiographic density, histologic quality, and mechanical strength of fusion at 12 weeks post-treatment rank consistently within the treatment groups. Optimal formulations are shown to perform similar to autograft in both the rate and strength of fusion. Fusion rates as high as 80% are observed within specific matrix/growth factor formulations. The average biomechanical strength of treated motion segments in the most efficacious formulation is 82% higher than that obtained with autograft, although this difference is not statistically significant. The fusion mass formed in response to matrix/growth factor formulations is composed of normal trabecular bone with a thin outer cortical plate and modest hematopoietic bone marrow. These results demonstrate that the combination of a mineralized collagen matrix with recombinant human growth and differentiation factor-5 maximizes the inherent conductive and inductive properties of each component, respectively, to provide an effective alternative to autograft for bone grafting procedures.

Inductive activity of recombinant human growth and differentiation factor-5.

Growth and differentiation factor-5 (GDF-5) is a divergent member of the transforming growth factor-beta/bone morphogenetic protein (BMP) superfamily that is required for proper skeletal patterning and development in the vertebrate limb. Based on the homology of GDF-5 with other bone-inducing BMP family members, the inductive activity of a recombinant form of human GDF-5 (rhGDF-5) was evaluated in a series of in vitro assays and in vivo bone-formation models. The in vitro response to rhGDF-5 resulted in the formation of chondrogenic nodules in fetal rat calvarial cells cultured in the context of collagen or collagen/hyaluronate extracellular matrices. Matrices loaded with rhGDF-5 induced ectopic cartilaginous and osseous tissue when implanted in subcutaneous or intramuscular sites. In non-human primate long-bone-defect and spinal-fusion models, rhGDF-5 combined with a mineralized collagen matrix induced bone formation in a manner equivalent to autogenous bone. These results highlight the unique potential of rhGDF-5 in a wide variety of orthopaedic applications.

Novel formulation of fibroblast growth factor-2 in a hyaluronan gel accelerates fracture healing in nonhuman primates.

Recent advances in understanding the biology of fracture healing and the availability of specific macromolecules has resulted in the development of novel treatments for injuries to bone. Fibroblast growth factor-2 or basic fibroblast growth factor (4 mg/ml), a potent mitogen, and hyaluronan (20 mg/ml), an extracellular matrix component, were combined into a viscous gel formulation intended for direct, percutaneous injection into fresh fractures. In an experimental primate fracture model, a bilateral 1-mm-gap osteotomy was surgically created in the fibulae of baboons. A single direct administration of this hyaluronan/fibroblast growth factor-2 formulation to the defect site significantly promoted local fracture healing as evidenced by increased callus formation and mechanical strength. Radiographic analysis showed that the callus area was statistically significantly larger at the treated sites than at the untreated sites. Specimens treated with 0.1, 0.25, and 0.75 ml hyaluronan/fibroblast growth factor-2 demonstrated a 48, 50, and 34% greater average load at failure and an 82, 104, and 66% greater energy to failure than the untreated controls, respectively. By histologic analysis, the callus size, periosteal reaction, vascularity, and cellularity were consistently more pronounced in the treated osteotomies than in the untreated controls. These results suggest that hyaluronan/fibroblast growth factor-2 may provide a significant advance in the treatment of fractures.

An osteoconductive collagen/hyaluronate matrix for bone regeneration.

A new type of collagen-hyaluronate (COL/HA) matrix was synthesized by cross-linking collagen fibers with modified hyaluronate polymers bearing active formyl groups. The resulting matrix is a three-dimensional scaffold consisting of interconnected pores with an average size of 40 microm and a high pore volume/surface area ratio. The covalent nature of the bond between the collagen fibers and the modified hyaluronate was demonstrated by extended elution with phosphate buffered saline and by extraction in increasing ionic gradients. The fraction of covalently bound hyaluronate in the matrix ranged from 5 to 25 w%. The total hyaluronate content of the COL/HA matrix affected both the in vitro non-enzymatic and enzymatic degradation as well as the in vivo turnover. When implanted in cranial defects in rats, the COL/HA matrix demonstrated good biocompatibility and exhibited greater osteoconductive potential than matrices composed of either cross-linked collagen or cross-linked hyaluronate alone.

Potential role of fibroblast growth factor in enhancement of fracture healing.

Fibroblast growth factors are present in significant amounts in bone and several studies have suggested that they may be involved in normal fracture healing. It is well established that fibroblast growth factors have mitogenic and angiogenic activity on mesoderm and neuroectoderm derived cells. Of particular interest as a member of the fibroblast growth factor family, basic fibroblast growth factor stimulates mitogenesis, chemotaxis, differentiation, and angiogenesis. It also plays an important role in the development of vascular, nervous, and skeletal systems, promotes the maintenance and survival of certain tissues, and stimulates wound healing and tissue repair. Animal studies have shown that the direct injection of fibroblast growth factor into fresh fractures stimulates callus formation, which provides mechanical stability to the fracture, accelerates healing, and restores competence. The matrix used to present the fibroblast growth factor at the fracture site plays a critical role in the effectiveness of the treatment. The evaluation of injectable basic fibroblast growth factor in a sodium hyaluronate gel for its effectiveness in stimulating fracture healing is described. When applied directly into a freshly created fracture in the rabbit fibula, a single injection of the basic fibroblast growth factor and hyaluronan results in the stimulation of callus formation, increased bone formation, and earlier restoration of mechanical strength at the fracture site. The hyaluronan gel serves as a reservoir that sequesters the basic fibroblast growth factor at the injection site for the length of time necessary to create an environment conducive to fracture healing. It is concluded that basic fibroblast growth factor and sodium hyaluronate act synergistically to accelerate fracture healing and that the combination is suitable for clinical evaluation as a therapy in fracture treatment.

Spreading and focal contact formation of human melanoma cells in response to the stimulation of both melanoma-associated proteoglycan (NG2) and alpha 4 beta 1 integrin.

In this study, we evaluated the potential role for a specific melanoma-associated chondroitin sulfate proteoglycan core protein, termed NG2, to collaborate with alpha 4 beta 1 integrin in focal contact formation in human melanoma cells. Although melanoma cells adhered to substrata coated with either the alpha 4 beta 1 integrin binding fibronectin synthetic peptide CS1-OVA or anti-NG2 mAbs, no spreading or focal contact formation was observed on either substratum. However, melanoma cells spread and formed focal contacts on "chimeric substrata" coated with CS1-OVA and the anti-NG2 mAb, 9.2.27, indicating that engaging both adhesion receptors changes the adhesion phenotype of melanoma cells by reorganizing the cytoskeleton. The collaboration between the two receptors is specific to fibronectin, since cells adherent on substrata coated with low concentrations of either laminin and 9.2.27 or type IV collagen and 9.2.27 failed to spread, while cells adherent on low concentrations of fibronectin and 9.2.27 exhibited a fully spread morphology. Two selective tyrosine kinase inhibitors, genistein and herbimycin A, totally inhibited cell spreading on the substrata coated with CS1-OVA and 9.2.27, indicating that tyrosine kinase(s) is important for cell spreading and focal contact formation. When cells were cultured on substrata coated with CS1-OVA and 9.2.27, two proteins (M(r) 130,000 and 120,000) were tyrosine phosphorylated in a genistein- and herbimycin A-sensitive fashion. These proteins were not immunologically related to pp125FAK or alpha 4 beta 1 integrin. Importantly, when melanoma cells were cultured on substrata coated with CS1 and then stimulated with 9.2.27-conjugated microsphere beads, formation of focal contacts and stress fibers was also observed, indicating that NG2 can collaborate with alpha 4 beta 1 integrin when each receptor is engaged on distinct and separate substrata. These results demonstrate that NG2 acts as a coreceptor for spreading and focal contact formation in association with alpha 4 beta 1 integrin in melanoma cells and suggest a model in which the NG2 core protein communicates to alpha 4 beta 1 integrin by an inside-out signaling mechanism.

Identification of a novel glycosaminoglycan core-like molecule. II. Alpha-GalNAc-capped xylosides can be made by many cell types.

The accompanying article (Manzi, A., Salimath, P. V., Spiro, R. C., Keifer, P. A., and Freeze, H. H. (1995) J. Biol. Chem. 270, 9154-9163) reported the complete structure of a novel molecule made by human melanoma cells incubated with 1 mM 4-methylumbelliferyl-beta Xyl (Xyl beta MU). The product resembles a common pentasaccharide core region found in chondroitin/dermatan sulfate glycosaminoglycans, except that a terminal alpha-Gal-NAc residue is found in a location normally occupied by beta-GalNAc in these chains or alpha-GlcNAc in heparan sulfate chains. In this paper we show that several other human cancer cell lines and Chinese hamster ovary cells also make alpha-GalNAc-capped xylosides. The [6-3H]galactose-labeled Xyl beta MU product binds to immobilized alpha-GalNAc-specific lectin from Helix pomatia and the binding is competed by GalNAc, but not by Glc. Binding to the lectin is destroyed by digestion with alpha-N-acetylgalactosaminidase, but not beta-hexosaminidase. The nature of the aglycone influences the amount and relative proportion of this material made, with p-nitrophenyl-beta-xyloside being a better promoter of alpha-GalNAc-terminated product than Xyl beta MU. This novel oligosaccharide accounts for 45-65% of xyloside-based products made by both human melanoma and Chinese hamster ovary cells when they are incubated with 30 microM Xyl beta MU, but at 1 mM both the total amount and the proportion decreases to only 5-10%. In both cell lines this product is replaced by a corresponding amount of Sia alpha 2,3Gal beta 4Xyl beta MU. Preferential synthesis of the alpha-GalNAc-capped material at very low xyloside concentration argues that it is a normal biosynthetic product and not an experimental artifact. This pentasaccharide may be a previously unrecognized intermediate in glycosaminoglycan chain biosynthesis. Since this alpha-GalNAc residue occurs at a position that determines whether chondroitin or heparan chains are added to the acceptor, it may influence the timing, type, and extent of further chain elongation.

Identification of a novel glycosaminoglycan core-like molecule. I. 500 MHz 1H NMR analysis using a nano-NMR probe indicates the presence of a terminal alpha-GalNAc residue capping 4-methylumbelliferyl-beta-D-xylosides.

beta-Xylosides compete with endogenous proteoglycan core proteins and act as alternate acceptors for synthesizing protein-free glycosaminoglycan chains. Their assembly on these alternate acceptors utilizes the same glycosyltransferases that make the protein-bound chains. Most studies using alternate acceptors focus on the production of sulfated glycosaminoglycan chains that are thought to be the major products. However, we previously showed that labeling melanoma cells with [6-3H]galactose in the presence of 4-methylumbelliferyl (MU) or p-nitrophenyl (pNP) beta-xylosides led to the synthesis of mostly di- to tetrasaccharide products including incomplete core structures. We have solved the structure of one of the previously unidentified products as, GalNAc alpha(1,4)GlcA beta(1,3)Gal beta(1,3)Gal beta(1,4)Xyl beta MU, based on compositional analysis by high performance liquid chromatography, fast atom bombardment, electrospray mass spectrometry, and one-dimensional and two-dimensional 1H NMR spectroscopy. The novel aspect of this molecule is the presence of a terminal alpha-Gal-NAc residue at a position that is normally occupied by beta-GalNAc in chondroitin/dermatan sulfate or by alpha-Glc-NAc in heparin or heparan sulfate chains. An alpha-GalNAc residue at this critical location may prevent further chain extension or influence the type of chain subsequently added to the common tetrasaccharide core.

Uncoupling of chondroitin sulfate glycosaminoglycan synthesis by brefeldin A.

Brefeldin A has dramatic, well-documented, effects on the structural and functional organization of the Golgi complex. We have examined the effects of brefeldin A (BFA) on the Golgi-localized synthesis and addition of chondroitin sulfate glycosaminoglycan carbohydrate side chains. BFA caused a dose-dependent inhibition of chondroitin sulfate glycosaminoglycan elongation and sulfation onto the core proteins of the melanoma-associated proteoglycan and the major histocompatibility complex class II-associated invariant chain. In the presence of BFA, the melanoma proteoglycan core protein was retained in the ER but still acquired complex, sialylated, N-linked oligosaccharides, as measured by digestion with endoglycosidase H and neuraminidase. The initiation of glycosaminoglycan synthesis was not affected by BFA, as shown by the incorporation of [6-3H]galactose into a protein-carbohydrate linkage region that was sensitive to beta-elimination. The ability of cells to use an exogenous acceptor, p-nitrophenyl-beta-D-xyloside, to elongate and sulfate core protein-free glycosaminoglycans, was completely inhibited by BFA. The effects of BFA were completely reversible in the absence of new protein synthesis. These experiments indicate that BFA effectively uncouples chondroitin sulfate glycosaminoglycan synthesis by segregating initiation reactions from elongation and sulfation events. Our findings support the proposal that glycosaminoglycan elongation and sulfation reactions are associated with the trans-Golgi network, a BFA-resistant, Golgi subcompartment.

Correlation of chondroitin sulfate proteoglycan expression on proliferating brain capillary endothelial cells with the malignant phenotype of astroglial cells.

Human glioblastomas (five of five), the most malignant astroglial-derived tumors, specifically express a chondroitin sulfate proteoglycan that is recognized by monoclonal antibody 9.2.27 and localized to the glioma cell surface, proliferating endothelial cells, and the perivascular extracellular matrix within the tumor bed. In contrast, the expression of this proteoglycan in normal adult neocortex and white matter is limited to the smooth muscle of small arteries, while normal glia, endothelial cells, and endothelial cell basement membranes are nonreactive. Moreover, two anaplastic astrocytomas, representing medium-grade astroglial-derived tumors, fail to react with monoclonal antibody 9.2.27. In culture, glioblastoma and capillary brain endothelial cells specifically synthesize a 250-kDa core protein and a high-molecular-mass chondroitin sulfate proteoglycan, recognized by monoclonal antibody 9.2.27. These data suggest a correlation between the expression of this chondroitin sulfate proteoglycan on proliferating brain capillary endothelial cells and the malignant phenotype of astroglial cells. The prominent perivascular localization of chondroitin sulfate proteoglycan makes it a marker for both proliferating brain capillary endothelial cells and the most malignant transformed astroglial cells, thus providing an ideal target for the immunotherapy of glioblastoma.

Post-translational processing of the glycoproteins of lymphocytic choriomeningitis virus.

Intracellular events in the synthesis, glycosylation, and transport of the lymphocytic choriomeningitis virus (LCMV) glycoproteins have been examined. We have shown by N-glycanase digestion that LCMV strain Arm-4 bears five oligosaccharides on GP-1 and two on GP-2. By pulse-chase labeling experiments in the presence of drugs which inhibit N-linked oligosaccharide addition and processing we demonstrate that addition of high mannose precursor oligosaccharides is necessary for transport and cleavage of the viral GP-C glycoprotein. Moreover, in the presence of tunicamycin which inhibits en bloc addition of these mannose-rich side chains, virus budding was substantially decreased and infectious virions were reduced by more than 1000-fold in the supernatant medium. Incubation in the presence of castantospermine, which permits addition of oligomannosyl-rich chains but blocks further processing, restored transport and cleavage of GP-C and maturation of virions. Finally, by temperature block experiments we have determined that maturation of GP-C oligosaccharides to an endoglycosidase H resistant form precedes cleavage to GP-1 and GP-2. The latter process is most likely to occur in the Golgi or post-Golgi compartment.

The invariant chain is a phosphorylated subunit of class II molecules.

The phosphorylation of the MHC, class II-associated invariant chain (gamma) is demonstrated in human B-lymphoblastoid, melanoma, and histiocytic lymphoma cell lines. Two-dimensional nonequilibrium gel electrophoresis of invariant chain and class II Ag immunoprecipitates isolated from [32P]orthophosphate-labeled cells demonstrates labeling of both free and class II-associated gamma, gamma s, and p41 forms of the invariant chain. The gamma 2/gamma 3 form of the invariant chain is not phosphorylated. Phosphoamino amino acid analysis of isolated invariant chain shows phosphorylation of serine residues. The isolation of invariant chain from 32P-labeled microsome preparations digested with proteinase K demonstrates that the phosphorylation occurs in the cytoplasmic tail. Limited proteolysis of [32P]orthophosphate-, [35S]cysteine-, and [35S]methionine-labeled invariant chain also indicates that the 32P-label is incorporated into the cytoplasmic domain. These results pinpoint serine residues at positions 9, 26, and 29 in the N-terminal cytoplasmic tail as potential sites for the phosphorylation of the invariant chain. Phosphorylation may be another mechanism by which the functions of invariant chain in class II-dependent immune responses are regulated.

Effect of inhibitors of N-linked oligosaccharide processing on the cell surface expression of a melanoma integrin.

The role of trimming and processing of N-linked oligosaccharides on the cell surface expression of the melanoma vitronectin receptor, a member of the integrin family of cell adhesion receptors, was examined by using specific glucosidase and mannosidase inhibitors. Inhibition of glucosidases I and II by castanospermine or N-methyldeoxynojirimycin delayed the vitronectin receptor alpha/beta chain heterodimer assembly and alpha chain cleavage and resulted in a decrease in the level of expression cell surface receptor. Conversely, the vitronectin receptor synthesized in the presence of the mannosidase I and II inhibitors, 1-deoxymannojirimycin and swainsonine, was transported normally to the cell surface with its alpha chain N-linked oligosaccharides in an endoglycosidase H-sensitive form. In the presence of swainsonine, time course studies of the cell surface replacement of control, endoglycosidase H-resistant receptor with an endoglycosidase H-sensitive form demonstrated a vitronectin receptor half-life of approximately 15-16 h. These studies provide evidence that the rates of assembly, proteolytic cleavage, and cell surface expression of the melanoma vitronectin receptor are dependent on the initial trimming of glucosyl residues from the alpha chain N-linked oligosaccharides.

Post-translational addition of chondroitin sulfate glycosaminoglycans. Role of N-linked oligosaccharide addition, trimming, and processing.

A melanoma proteoglycan model system has been used to examine the role of core protein asparagine-linked (N-linked) oligosaccharides in the transport and assembly of proteoglycan molecules. The use of agents which block discrete steps in the trimming and processing of core oligosaccharides (castanospermine, 1-deoxynojirimycin, N-methyldeoxynojirimycin, 1-deoxymannojirimycin, and swainsonine) demonstrates that removal of glucose residues from the N-linked oligosaccharides is required for the cell surface expression of a melanoma proteoglycan core protein and for the conversion of the core protein to a chondroitin sulfate proteoglycan. However, complete maturation of the oligosaccharides to a "complex" form is not required for these events. Treatment of M21 human melanoma cells with the glucosidase inhibitors castanospermine, 1-deoxynojirimycin, or N-methyldeoxynojirimycin results in a dose-dependent inhibition of glycosaminoglycan (GAG) addition to the melanoma antigen recognized by monoclonal antibody 9.2.27. In contrast, treatment with the mannosidase inhibitors 1-deoxymannojirimycin and swainsonine does not effect GAG addition. Identical results are obtained when the major histocompatibility complex class II antigen gamma chain proteoglycan is examined in inhibitor-treated melanoma and B-lymphoblastoid cells. These data, in conjunction with the known effects of the glucosidase and mannosidase inhibitors on the transport and secretion of other glycoproteins support the hypothesis that the addition, trimming, and processing of N-linked oligosaccharides is involved in the transport of certain proteoglycan core proteins to the site of GAG addition and to the cell surface.

A human brain glycoprotein related to the mouse cell adhesion molecule L1.

We have employed monoclonal antibody 5G3, an antibody used to label human tumor cells of neural origin (Mujoo, K., Spiro, R.C., and Reisfeld, R. A. (1986) J. Biol. Chem. 261, 10299-10305), to isolate and characterize a large glycoprotein from normal adult human brain. This protein was compared to mouse L1 (Rathjen, F. G., and Schachner, M. (1984) EMBO J. 3, 1-10), a neural cell surface glycoprotein implicated predominantly in neurite-neurite interactions. On the basis of the following results the 5G3 antigen is considered to be the human homologue of mouse L1. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, both proteins share similar molecular masses of their carbohydrate-depleted or undepleted components. In tryptic fingerprint analyses of the iodinated L1 and 5G3 components, 65% of the resolved peptides comigrated. Comparison of NH2-terminal amino acid sequences revealed a high degree of homology between human 5G3 and mouse L1, with 11 of 15 residues being identical. Furthermore, polyclonal antibodies to human 5G3 antigen were found to be cross-reactive with mouse L1 antigen and vice versa. All components of 5G3 and L1 antigens show considerable charge heterogeneity with partial overlapping of regions in isoelectric focusing followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. These findings provide a basis for studying the role of the human L1 homologue in human diseases.

Biosynthetic and functional properties of an Arg-Gly-Asp-directed receptor involved in human melanoma cell attachment to vitronectin, fibrinogen, and von Willebrand factor.

M21 human melanoma cells express an Arg-Gly-Asp-directed adhesion receptor composed of noncovalently associated alpha and beta chains. To establish the structural and functional properties of this receptor on M21 human melanoma cells, stable variant cell lines were selected that express altered alpha chain levels. One of these variants, M21-L, fails to synthesize alpha chain protein or its mRNA, yet does produce normal levels of the beta chain. In these cells the beta chain does not reach the cell surface but rather accumulates within the cell. M21-L cells lacking the alpha chain are incapable of attaching to vitronectin, von Willebrand factor, fibrinogen, or an Arg-Gly-Asp-containing heptapeptide yet attach normally to fibronectin, whereas the unselected M21 cells attach to all of these adhesive proteins. In addition, a monoclonal antibody, LM609 generated to a functional site on the intact receptor, is capable of preventing M21 cell attachment to vitronectin, von Willebrand factor, fibrinogen, and the Arg-Gly-Asp peptide but not to fibronectin. Following a 2-min biosynthetic pulse-label, the newly synthesized alpha chain remains in free form for 5 min and then associates with previously synthesized beta chain present in an intracellular pool. Once oligomerization takes place, the receptor gains the capacity to recognize Arg-Gly-Asp, and at this time the epitope recognized by monoclonal antibody LM609 is formed.

Characterization of a unique glycoprotein antigen expressed on the surface of human neuroblastoma cells.

In order to develop a molecular probe to delineate chemical and biological characteristics of human neuroblastoma cells, a murine monoclonal antibody (Mab 5G3) was produced that is directed to a glycoprotein, preferentially expressed on the surface of such cells. This antibody is of IgG2a isotype, has an association constant of 8 X 10(9) M-1, and reacts preferentially with human neuroblastoma cell lines and fresh frozen tissue sections in enzyme-linked immunosorbent assay and immunoperoxidase assays, respectively. Minimal reactivity is observed with a variety of lymphoblastoid cell lines and normal fetal and adult tissues. Mab 5G3 specifically recognizes a neuroblastoma target glycoprotein antigen of 215 kDa that is derived from a 200-kDa precursor, as evident from pulse-chase biosynthetic studies. Treatment with tunicamycin revealed that both molecules contain N-asparagine-linked oligosaccharides; however, only the 215-kDa species is resistant to treatment with endo-beta-N-acetylglucosaminidase H and sensitive to neuraminidase, indicating that it contains trimmed and terminally sialylated oligosaccharides of the "complex" type. In contrast, the 200-kDa precursor is sensitive to endo-beta-N-acetylglucosaminidase H and resistant to neuraminidase treatment indicating that it contains high-mannose non-processed oligosaccharides. The 215-kDa molecule is sulfated, phosphorylated at serine residues, and expressed on the cell surface. A molecule of 200 kDa is detected by Mab 5G3 in spent culture medium of human neuroblastoma cells which is neither sulfated nor phosphorylated.

Inhibition of post-translational modification and surface expression of a melanoma-associated chondroitin sulfate proteoglycan by diethylcarbamazine or ammonium chloride.

Cultured human melanoma M21 cells were treated with diethylcarbamazine (DEC), an inhibitor of proteoglycan biosynthesis in rat chondrosarcoma cells, to examine the assembly and transport of a chondroitin sulfate proteoglycan to the plasma membrane. Pretreatment of melanoma cells at 37 degrees C for 15 min with increasing doses of DEC followed by a 60-min pulse with [35S]sulfate in the presence of DEC resulted in a dose-related inhibition of incorporation of [35S]sulfate into macromolecules. In cells incubated for 75 min with both 1 mM beta-D-xyloside and 15 mM DEC, synthesis and secretion of beta-D-xyloside-bound 35S-glycosaminoglycans were inhibited by more than 80% as compared to cells treated with beta-D-xyloside alone; this inhibition was reversible. As assessed by [3H]serine incorporation into protein, overall protein synthesis was not substantially inhibited by DEC treatment. Detergent lysates from [35S]methionine-labeled melanoma cells were incubated with a monoclonal antibody (9.2.27) that specifically recognizes the peptide core of the melanoma proteoglycan. As assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the immunoprecipitate, a 240,000 Mr endoglycosidase H (Endo-H)-sensitive intermediate was the only form of the proteoglycan present inside the cells when the cultures were treated for 60-120 min with 10-15 mM DEC. When the melanoma cells were incubated for 10 min with 15 mM DEC and 100 mu Ci/ml of [35S]methionine, washed, and then chased for 15 min to 4 h in radioactive-free medium, the 240,000 Mr Endo-H-sensitive intermediate was slowly converted to a 250,000 Endo-H-resistant intermediate but not to a mature proteoglycan molecule that possessed chondroitin sulfate glycosaminoglycans. SDS-PAGE analysis of cell surface immunoprecipitates revealed that only a small amount of the 250,000 Mr intermediate was transported to the plasma membrane within 5 h of incubation in the presence of DEC. Proteoglycan synthesis was also inhibited when the melanoma cells were incubated for 60-120 min with ammonium chloride, but unlike DEC-treated cells the majority of the synthesized peptide core was converted to a 245,000 Mr Endo-H-resistant intermediate that was detected on the cell surface. Light and electron microscopic analysis of DEC-treated melanoma cells revealed large vacuoles and a distended Golgi and endoplasmic reticulum. Ammonium chloride-treated cells contained fewer vacuoles than DEC-treated cells but more vacuoles than normal cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Functional association of class II antigens with cell surface binding of Epstein-Barr virus.

A functional role of class II antigen in the binding of Epstein-Barr virus (EBV) was deduced from the study of membrane proteins on Jijoye, an EBV receptor (EBVR)-positive B cell line, and its mutant, EBVR-negative daughter cell line, P3HR-1. From gel electrophoresis of radiolabeled microsomal membrane proteins and immunoprecipitates, we identified class II antigen on Jijoye but not on P3HR-1 cells and the presence of Ii on both cell lines. The role of these molecules in EBVR function was tested by antibody blocking of virus adsorption. Anti-p23,30 serum (to class II antigen) was found to block binding of EBV to B lymphoblasts under conditions in which normal rabbit serum, rabbit antiserum to butyrate-treated P3HR-1 cells (with ample anti-Ii antibodies), and rabbit anti-p44,12 (to class I antigen and beta 2-microglobulin) serum did not block virus binding. Only one of four commercial monoclonal antibodies (MoAb) to framework epitopes on class II antigens blocked binding of EBV, whereas all four MoAb demonstrated immunofluorescent reactivity with the EBVR+ Raji cells. In previous studies of binding of EBV to hairy leukemic cells, a substantial subpopulation of HLA-DR+, EBVR- cells was identified, in addition to HLA-DR+, EBVR+ cells. These findings were consistent with the view that the HLA-DR complex has a role in the binding of EBV but that other components are also needed for the expression of EBVR function.