Radioimmunoassay of a cancer-related glycoprotein. Circulating levels

Circulating levels of (a) tumor-related glycoprotein(s) were determined by radioimmunoassay for a variety of patients and controls, and correlated with sialic acid concentration. Levels were highest in patients with metastatic disease and progressively declined to those with localized disease receiving therapy. Values for normal, adjuvant, and cured patients were significantly lower. Sialic acid concentrations correlated best for the metastatic group but not for the normals.     

Some further information about the abnormal membrane glycoprotein associated with malignancy.

The abnormal membrane glycoprotein that we previously found to be associated with malignancy in a wide range of different tumours is present in the cell membrane as a dimer and appears to have a specific binding site for glucose.     

Characterization of a membrane surface glycoprotein associated with T-cell activation

A cell surface antigen (gp140) was previously shown to exist on T cell subsets as well as on monocytes and macrophages in normal peripheral blood. Elevated expression of this antigen was associated with immune system disorders, acute lymphocytic leukemias, and in vitro activation of T cells. The antigen could be identified with monoclonal antibody (MAb) T305. Gp140 was a biosynthetic product of T cells because it could be labeled with [3H]leucine or [3H] glucosamine. Biochemical studies of gp140 used high performance liquid chromatography with nitrocellulose blotting to isolate aliquots suitable for 125I radiolabeling and immunoprecipitation to demonstrate: a) a reduction in m.w. of gp140 KD to 90 KD after deglycosylation by trifluoromethanesulfonic acid, b) alteration of isoelectric point from 4.1 to 5.7 after neuraminidase treatments, c) absence of N-linked sugars based on resistance to endoglycosidase F, d) resistance to trypsin and chymotrypsin digestion but susceptibility to pronase, and e) presence of sialic acid and lactosaminoglycan as O-linked sugars. Gp140 could be labeled with the periodate/NaB[3H]4 technique, indicating its similarity to a class of sialoglycoproteins previously described on activated T-cells in mouse and man. The antigenic epitope recognized by MAb T305 contains sialic acid linked (2----3) to galactose; however, periodate oxidation of the exocyclic ring of sialic acid did not affect binding by MAb T305. In an attempt to determine the functional role of gp140, we tested the ability of MAb T305 to block: a) proliferation of peripheral blood lymphocytes to mitogens, b) response to interleukin 2 (IL 2) of an IL 2 dependent T cell line, and c) growth of a T-ALL derived cell line. No inhibition of proliferation or growth was noted. Although the function of gp140 remains unknown, its association with lymphocyte activation and certain disease states suggests that it may provide a target for modulation of the immune response. These studies characterize the structural features of gp140 and further define the epitope recognized by MAb T305.     

Radioimmunoassay of a bovine pregnancy-associated glycoprotein in serum: its application for pregnancy diagnosis.

A sensitive and specific double-antibody RIA for a bovine pregnancy-associated glycoprotein (bPAG) is described. The limit of detection was 0.2 ng/ml. The assay was specific for bPAG in that pituitary and placental gonadotropic hormones and other placental or serum proteins assayed in serial dilutions did not cross-react. The RIA allowed measurement of bPAG in placental extracts, fetal serum, fetal fluids, and serum or plasma of pregnant cows. About 20% of unbred heifers and nonpregnant cows had detectable levels ranging from 0.30 +/- 0.09 to 0.50 +/- 0.17 ng/ml (mean +/- SD), and 15% of bull sera showed higher concentrations (3.01 +/- 1.73 ng/ml) of bPAG or bPAG-like protein. Variations among animals was observed in fetal serum bPAG concentrations. Bovine PAG was detected in maternal peripheral blood at Day 22 of pregnancy (mean +/- SD, 0.38 +/- 0.13 ng/ml) in some animals and at Day 30 in all pregnant cows. Peripheral serum bPAG levels increased progressively to 3.60 +/- 1.73 ng/ml (mean +/- SD) at Day 30 of pregnancy, to 24.53 +/- 8.81 ng/ml at Day 120, and to 1551.91 +/- 589.68 ng/ml at Day 270. Peak concentration of bPAG was 2462.42 +/- 1017.88 ng/ml and it occurred 1-5 days prior to parturition. After delivery, bPAG concentrations decreased steadily to 499.63 +/- 267.20 ng/ml at Day 14 postpartum (pp), 10.12 +/- 7.84 ng/ml at Day 60 pp, and 1.44 +/- 1.08 ng/ml at Day 90 pp. The undetectable concentration (less than 0.20 ng/ml) was reached by Day 100 +/- 20 pp. An investigation undertaken in Holstein heifers, Holstein cows, and Hereford cows used as recipients for purebred Holstein embryos supplied evidence of the influence of breed of recipient and sex of fetuses on peripheral concentrations of bPAG. A herd of 430 Holstein-Friesian heifers that had received transferred embryos were bled at Day 35 postestrus (pe) for measurement of bPAG. The bPAG was detected in 287 of 430 serum samples analyzed. By rectal palpation performed at Day 45 pe, 267 heifers with detectable levels of bPAG at Day 35 pe were confirmed to be pregnant as were 3 of 143 heifers previously diagnosed as not pregnant by RIA. These results suggest that detection of this placental-specific antigen in the serum could be used as a specific serological method for early pregnancy diagnosis in cattle from 28 days after breeding.     

Intracellular lectins associated with N-linked glycoprotein traffic

The vectorial intracellular transport of N-glycan-linked glycoproteins is indispensable for biological functions. In order to sort these glycoproteins to the correct destination, animal intracellular lectins play important roles as sorting receptors. The roles of such lectins in the biosynthetic pathway from the endoplasmic reticulum (ER) to the cell surface are addressed in this review. Calnexin and calreticulin function via specific carbohydrates in quality control of newly synthesized glycoproteins in the ER, and ERGIC-53 seems to function in the transport of glycoproteins from ER to the Golgi complex. In addition to the well-understood role of mannose 6-phosphate receptor in lysosomal protein sorting, the vesicular integral protein of 36 kDa (VIP36) functions as a sorting receptor by recognizing high-mannose type glycans containing alpha1-->2Man residues for transport from Golgi to the cell surface in polarized epithelial cells.     

Characterization of a cell surface glycoprotein associated with maturation of rat spermatozoa

The principal galactose oxidase/NaB[³H]₄-labeled membrane protein of rat caudal epididymal spermatozoa was isolated by hydrophobic interaction chromatography. The protein is released from the membrane by the action of phosphatidylinositol specific phospholipase C, and thereby its properties are transformed from those of a protein anchored to the hydrophobic membrane to those of a hydrophilic solution protein. Because it is the only membrane-associated protein released by the enzyme which did not absorb to a propylaspartate resin, a simple, single step purification procedure was devised. Although the amino terminus of the protein is blocked to Edman degradation, the majority of the protein structure was determined from a series of tryptic peptides and from limited acid hydrolysis. Approximately 65% of the protein mass is carbohydrate which is primarily attached through O-glycosidic bonds to the 18 threonines. The molecular weight of the glycoprotein was estimated to be 16,600, considerably smaller than the M_r = 26,000 to 37,000 previously determined by gel electrophoresis. The anomalous electrophoretic behavior is undoubtedly due to the large percentage of carbohydrate. The distribution of carbohydrate on the protein side chains suggests the protein may form a positively charged, specialized scaffolding for the presentation of the carbohydrate moieties. Because the appearance of the ability to label the protein with galactose oxidase is correlated with sperm maturation in the epididymis, the glycoprotein structures may be an important componetn in the fertilization process. The combination of linkage by glycosylphosphatidylinositol and low molecular weight mucin-like structure indicates this may be a member of a new class of membrane proteins. © 1994 Wiley-Liss, Inc.     

Demonstration of glycoprotein antigens associated with stomach tumors

After precipitation of the glycoproteins from gastric tumor tissues by caprylic acid, immunochemical methods were applied to the research of specific gastric tumor antigens; this precipitation process by caprylic acid was less aggressive than the use of urea or proteases. The purification of a fraction associated with tumor tissue has been carried out by affinity chromatography with a specific antiserum immobilized on Sepharose. The obtained fraction contains 4 proteic antigens. One of them is common with all the gastric extracts. Neither CEA, nor alpha-foetoprotein has been detected in this fraction. The corresponding antiserum seems to show a tumor specificity, whereas tissue specificity has to be demonstrated.     

Radioimmunoassay of the link proteins associated with bovine nasal cartilage proteoglycan.

Link proteins from bovine nasal cartilage have been purified by preparative polyacrylamide gel electrophoresis in sodium dodecyl sulfate (Baker, J.R., and Caterson, B. (1979) J. Biol. Chem. 254, 2387-2393) and used to raise antisera in rabbits. A sensitive radioimmunoassay procedure utilizing binding of 125I-labeled antigen . antibody complexes to Protein A of Staphylococcus aureus has served to demonstrate the specificity of the antisera for the link proteins. The lack of reactivity with proteoglycan fractions indicates that link proteins and proteoglycan do not share antigenic determinants. This result is in accord with published cyanogen bromide peptide cleavage data (Baker, J.R., and Caterson B. (1977) Biochem. Biophys. Res. Commun. 77, 1-10) which showed proteoglycan and link protein to be structurally dissimilar. The radioimmunoassay procedure has been used to quantitate small amounts of link protein which remain associated with proteoglycan after purification by equilibrium density gradient centrifugation in 4 M guanidine HCl and by gel chromatography in sodium dodecyl sulfate.     

A neuronal surface glycoprotein associated with the cytoskeleton

A cytoskeleton-associated glycoprotein of 130-kilodalton molecular mass (GP 130) was purified from a nonionic detergent-insoluble fraction of 10-16-d-old chicken embryo brains. GP 130 is tightly associated with other proteins in actin-containing complexes (Moss, D.J., 1983, Eur. J. Biochem., 135:291-297); thus, pure protein preparations were obtained only after the partial dissociation of the complexes with the zwitterionic detergent, dimethyl dodecyl glycine (EMPIGEN BB), followed by ion-exchange chromatography and electrophoresis on preparative SDS polyacrylamide gels. Specific monoclonal and polyclonal antibodies were raised to GP 130 and used to examine its distribution in the developing nervous system. Experiments with these antibodies revealed that GP 130 is confined to nervous tissue and is restricted to the surface of neurons in cultures derived from both the central and peripheral nervous systems. This novel glycoprotein is immunologically unrelated to the neuronal cell adhesion molecule (N-CAM), or to vinculin, a protein of similar molecular mass which has been suggested to link actin filaments to the plasma membrane. In the developing chicken embryo brain, GP 130 is first detectable around day 8 after fertilization and increases to approximately 50% of its adult level by embryonal day 13. In contrast, no increase is observed over a similar developmental period in sciatic nerve. In the adult chicken, GP 130 is most abundant in brain and has a particularly high content in areas rich in dendrites and synapses.     

Enzymatic sulfation of a large molecular weight glycoprotein associated with pig brain membranes

Pig brain membranes catalyze the transfer of [³⁵S]sulfate from 3′-phosphoadenosine 5′-phospho[³⁵S]sulfate into two macromolecular endogenous acceptors. Several operational enzymatic properties of the sulfotransferase activity have been defined. An apparent K_m = 0.65 μm for 3′-phosphoadenosine 5′-phosphosulfate has been determined for the pig brain in vitro sulfotransferase system. Direct proof for the absolute requirement of the 3′-phosphate moiety of 3′-phosphoadenosine 5′-phosphosulfate is presented. The nucleotide end product, 3′,5′-ADP, is a potent competitive inhibitor of the pig brain sulfotransferase activity. One of the major products enzymatically labeled during incubation with 3′-phosphoadenosine 5′-phospho[³⁵S]sulfate is a membrane-bound glycoprotein of high molecular weight. The sulfated glycoprotein appears to be an integral membrane glycoprotein, requiring 1% Triton X-100 for extraction. An ³⁵S-labeled oligosaccharide, released by mild base treatment, contains O-sulfate ester groups and at least one N-acetylneuraminic acid residue. The sulfated glycoprotein has an apparent molecular weight of 198,000. Under the same in vitro conditions [³⁵S]sulfate is also incorporated into a membrane-associated ³⁵S-labeled proteoglycan having the properties of heparan sulfate. The ³⁵S-labeled proteoglycan is electrostatically bound to the pig brain membranes, and can be readily extracted with 1 m NaCl.     

S1 nuclease of Aspergillus oryzae: a glycoprotein with an associated nucleotidase activity

S₁ nuclease (EC 3.1.30.1) of Aspergillus oryzae has been purified 1600-fold by a procedure designed to remove traces of contaminating phosphatases. The nearly homogeneous enzyme was found to be a glycoprotein with a carbohydrate content of 18%. At pH 4.5 the enzyme preparation hydrolyzed single-stranded DNA, RNA, 3′-AMP, and 2′-AMP at relative rates of 100, 52, 13, and 0.05, respectively. The 3′-nucleotidase activity of this single-strand specific nuclease is inhibited by single-stranded DNA but not by double-stranded DNA. Three forms of the enzyme, with isoelectric points of 3.35, 3.53, and 3.67, were observed on electrofocusing, and each form exhibited the same relative activity on single-stranded DNA and 3′-AMP. Enzymatic hydrolysis of nucleotides occurred over a broad range of pH, with maximal activity at pH 6–7. Ribonucleotides were hydrolyzed approximately 100-fold more rapidly than deoxyribonucleotides. A high degree of base specificity was not observed. The 3′-nucleotidase activity was stimulated by Zn²⁺, but not by other divalent cations tested.     

Intracellular processes associated with glycoprotein transport and processing.

The intracellular transport of mucus glycoprotein precursor (apomucin) from endoplasmic reticulum (ER) to Golgi was quantitated by the immunoprecipitation with 3G12 antimucin monoclonal antibody and by estimation of the apomucin glycosylation using UDP-[3H]galactose. The assembly of the entities carrying apomucin to Golgi was assessed by electron microscopy and by quantitation of the incorporation of [14C]choline, [14C]ethanolamine, and [14C]oleic acid into their lipids. The microscopic image of the isolated transport components revealed a population of 80- to 100-nm vesicles with occasional membranes of the ER used for their synthesis. On the average, the vesicles contained 82 ng apomucin/microgram of protein and 80-90% of the total incorporated lipid precursors. From that, 91% of [14C]choline was detected in phosphatidylcholine, and 9% in phosphatidylethanolamine, lysophosphatidylcholine, and sphingomyelin. With [14C]oleate, 54% of the label was incorporated into ceramide, diglyceride, and phosphatidic acid, 35% to phosphatidylcholine, 7% in phosphatidylethanolamine, and 2% in sphingomyelin. After incubation of the vesicles with Golgi, the apomucin was found glycosylated and the lipids of the transport vesicles incorporated into Golgi membranes. The fusion of the vesicular membranes was accompanied by the synthesis of sphingomyelin. In the Golgi, 39-55% of the radiolabeled phosphatidylcholine of transport vesicles was converted to sphingomyelin. The results indicate that the newly synthesized membranes of apomucin transporting vesicles are enriched in phosphoglycerides and ceramides. Upon fusion with the Golgi, the membranes of the vesicles are replenished with sphingomyelin by exchange reaction between phosphatidylcholine and ceramide.     

Immunoexpression of tenascin as a predictor of the malignancy potential of oral leukoplakia associated with a tobacco habit

Oral leukoplakia is a morphological alteration of tissue that is an early indicator for malignancy. Tenascin (TN) is a large hexameric extracellular matrix (ECM) protein with anti-adhesive properties that fosters cell migration during development, wound healing and tissue remodeling; it is present in small amounts in adult tissues. Overexpression of TN in a pathological condition may be either a cause or a consequence of the disease. We evaluated the efficacy of TN for early prediction of tobacco-associated oral cancers. We studied retrospectively 95 cases of oral leukoplakia, including mild, moderate and severe cases, using immunohistochemistry for TN. We evaluated the intensity, area and pattern of TN expression. Greater intensity and area of TN expression was observed in mild and severe dysplasia than in moderate dysplasia. Most cases showed a reticular pattern of expression, especially in mild and moderate dysplasia; a fibrillar pattern was more evident in severe dysplasia. We also observed homogeneous expression pattern in some cases. TN is a marker for dysplastic changes in epithelium and its expression may be helpful for predicting the malignancy potential of tobacco-associated oral leukoplakia.     

Characterisation of a soluble trypsin fragment of GP130: a neuronal glycoprotein associated with the cytoskeleton

A neuronal glycoprotein (GP130) that is associated with the cytoskeleton [Ranscht et al: J Cell Biol 99:1803-1813, 1984] remains insoluble in 0.1 M NaOH, a property typical of integral membrane proteins. At present it is possible to solubilise and hence isolate GP130 only under denaturing conditions. However, a large fragment of apparent molecular weight 120K is released into solution by trypsin. The fragment corresponds to the extracellular region of the glycoprotein as shown by the fact that it is released from live cultures of chicken sympathetic neurons and by its retention of concanavalin A-binding activity. The soluble extracellular fragment has been purified using mild biochemical techniques, which are expected to retain its biological activity. Measurement of the sedimentation coefficient, Stokes radius, and frictional ratio in addition to metal shadowing of the fragment show that it has a molecular weight of about 120K and is asymmetric, probably rod-shaped with a long axis of more than 20 nm.     

Treating rheumatic patients with a malignancy

Management of patients with inflammatory rheumatic disease and a history of (or even a current) malignant disease poses some particular challenges. As direct evidence of the risk of (recurrent or de novo) malignancy in patients with a history of malignant disease is scarce, such a risk may be estimated indirectly from the principal carcinogenicity of the respective drug to be used or (also indirectly) from cancer reactivation data from the transplant literature. In general, cancer risk is increased in patients receiving combination immunosuppressive treatment, but the risk in patients receiving individual drugs (with the exception of alkylating agents) remains entirely unclear. Indirect evidence supports the intuitive concept that the risk of cancer decreases over time after a successful cancer treatment. The only two studies in rheumatic patients with a cancer history were small and have not been able to show an increase in cancer reactivation. The risk of reactivation also depends on the site and location of the prior malignancy. In conclusion, the decision to treat a patient with a history of cancer immunosuppressively should be shared by the rheumatologist and the oncologist. Once the decision is established, such patients need intensive and close monitoring.     

Undulin, an extracellular matrix glycoprotein associated with collagen fibrils

Undulin, a novel noncollagenous extracellular matrix protein, was isolated from skin and placenta. In polyacrylamide gels most of the unreduced protein migrates with Mr above 1,000,000 yielding bands A (Mr 270,000), B1 (Mr 190,000), and B2 (Mr 180,000) after reduction. Undulin is biochemically and immunochemically distinct from other previously characterized large matrix glycoproteins. Immunoblotting using monoclonal antibodies suggests that bands A and B are closely related. Electron microscopy reveals undulin as structures consisting of an approximately 80-nm-long-tail with a nodule on one end and with one or two shorter arms on the other. Ultrastructurally immunolabeled undulin is found mainly between densely packed mature collagen fibrils. Indirect immunofluorescence shows bundles of uniform wavy fibers in dense connective tissues superimposable on a subpopulation of type I collagen structures. This suggests that undulin serves a specific yet unknown function in the supramolecular organization of collagen fibrils in soft tissues.     

Characterization of an oviductal glycoprotein associated with the ovulated hamster oocyte

Hamster oviducts in culture incorporate [35S]-methionine into secretory proteins. One of these proteins is immunoprecipitated by a monoclonal antibody specific to an antigen found in oviductal oocytes but not in ovarian oocytes. This antigen, called oviductin, is progressively added to the oocyte during its transit through the oviduct. Oviductin migrates as a diffuse band with a molecular mass between 160 and 250 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. The electrophoretic behavior of this protein suggests the presence of polysaccharide side chains. Chemical deglycosylation causes a decrease in molecular mass and removes the antigenic determinant originally present on the glycoprotein. By using the radiation inactivation method, the molecular mass of the core protein has been found to be approximately 44 kDa. These results indicate that the oviduct is an actual site of synthesis of the oviductin. This glycoprotein contains a high proportion of sugar residues, which account for antigenic determinant recognized by the monoclonal antibody.     

Phosphorylation of the multidrug resistance associated glycoprotein

Drug-resistant cell lines derived from the mouse macrophage-like cell line J774.2 express the multidrug resistance phenotype which includes the overexpression of a membrane glycoprotein (130-140 kilodaltons). Phosphorylation of this resistant-specific glycoprotein (P-glycoprotein) in intact cells and in cell-free membrane fractions has been studied. The phosphorylated glycoprotein can be immunoprecipitated by a rabbit polyclonal antibody specific for the glycoprotein. Phosphorylation studies done with partially purified membrane fractions derived from colchicine-resistant cells indicated that (a) phosphorylation of the glycoprotein in 1 mM MgCl2 was enhanced a minimum of 2-fold by 10 microM cAMP and (b) the purified catalytic subunit of the cAMP-dependent protein kinase (protein kinase A) phosphorylated partially purified glycoprotein that was not phosphorylated by [gamma-32P]ATP alone, suggesting that autophosphorylation was not involved. These results indicate that the glycoprotein is a phosphoprotein and that at least one of the kinases responsible for its phosphorylation is a membrane-associated protein kinase A. The state of phosphorylation of the glycoprotein, which is a major component of the multidrug resistance phenotype, may be related to the role of the glycoprotein in maintaining drug resistance.