The influence of alpha1-acid glycoprotein (orosomucoid) and its glycoforms on the function of human thyrocytes and CHO cells transfected with the human TSH receptor.

Local immunological reactions might influence the structure of alpha1-acid glycoprotein (AGP, orosomucoid) leading to a pathological condition in e.g. the thyroid. The aim of this study was to investigate whether the AGP molecule had a direct effect on thyroid cell function in vitro. The influence of AGP and its three glycoforms, TSH (1.0 U/l), serum samples and several sugars (methyl-mannose, methyl-glycoside, N-acetyl-D-galactose, N-acetyl-D-glycoside, neuramidase) were studied with respect to their influence on the function of the Chinese Hamster Ovary (CHO) cell line transfected with the human TSH receptor (hTSHr) and on human thyroid follicular epithelial cells (TFEC) in secondary cultures. We found that low concentrations of AGP (0.001-0.05 microg/l) stimulated while high concentrations of AGP (0.25-1.0 microg/l) inhibited cAMP accumulation in both cell systems (n=24, P<0.0002). In CHO cells (JP26) and TFEC glycoforms 1 (n=9), 2 (n=12) or 3 (n=11) significantly inhibited the TSH stimulated cAMP production, respectively, compared to controls (P<0.0001) and was partially reversed by mannose (P<0.0004). Control CHO cells (JP02) without the hTSHr showed no response. The specificity of the reaction was further confirmed by binding of biotinylated glycoforms and streptavidin conjugated FITC to both cell systems. This is the first report demonstrating that AGP and/or its glycoforms affects thyroid cell function in vitro and that it does so by influencing the second messenger cAMP probably by interacting directly with the TSH receptor.

Long-term pathologic changes of alpha1-acid glycoprotein (orosomucoid) glycoforms in autoimmune thyroid disease.

OBJECTIVE: We measured alpha1-acid-glycoprotein (AGP) in patients with autoimmune thyroid disease to study a possible relationship between microheterogeneity of the naturally occurring glycoforms of AGP and autoimmune thyroid disease. DESIGN, PATIENTS, MEASUREMENTS: In a group of 12 fasting thyrotoxic patients (11 females, mean age: 43 years) with newly diagnosed Graves' disease (subgroup 1), we measured serum concentrations of total AGP and its 3 glycoforms (micromol/l, crossed affinity immunoelectrophoresis with con A in the first dimension gel) as well as total thyroxine, total triiodothyronine, thyrotropine, thyroid peroxidase antibodies (anti-TPO), antibodies against the TSH receptor (TRAb, TRAK), at baseline and after 12 months of antithyroid drug therapy (ATD). For comparison, 4 subgroups of thyroid patients (patients with Graves' disease and thyroid associated ophthalmopathy (TAO) (subgroup 2, n = 10), radioiodine treated Graves' patients (subgroup 3, n = 7), Graves' patients without TAO (subgroup 4, n = 13), patients with Hashimoto's thyroiditis (subgroup 5, n = 8)) and 25 normal controls (17 females, mean age: 38 years) were studied. RESULTS: In subgroups of TRAb positive Graves patients' serum levels of glycoform 1, 2 or 3 increased significantly (p < 0.005) after 12 months of ATD as compared to both baseline of that person or normal controls. No significant changes were found in the TRAb negative Hashimoto subgroup. CONCLUSION: Patients with autoimmune Graves' disease changed their relationship to AGP, and thus a role of AGP and its 3 glycoforms is suggested in the pathogenesis of Graves' disease.

Insulin/FGF-binding ciliary membrane glycoprotein from Tetrahymena.

Triton X-100 extracted ciliary membrane protein from isolated cilia, prepared from the protozoon Tetrahymena thermophila, were fractionated by affinity chromatography on columns with covalently bound fibroblast growth factor (FGF), insulin, or concanavalin A (ConA), respectively. The eluted proteins were further analyzed by electrophoresis on sodium dodecyl sulfate polyacrylamide gels, isoelectric focusing, and by immunoblotting techniques using antibodies against the FGF receptor, platetelet derived growth factor (PDGF) receptor alpha-subunit, and insulin receptor beta-subunit. The particular antibodies were chosen because the peptides PDGF, FGF, insulin, and ConA are chemoattractants in this organism and corresponding binding (receptor) proteins could be expected to be identified. A 66 kDa protein fraction was eluted from the FGF-MiniLeak agarose, insulin-MiniLeak agarose and ConA sepharose. This fraction responded in Western immunoblots to an antibody against the beta-subunit of the human insulin receptor, to an antibody against the PDGF receptor (PDGFR) and also to an antibody against the bovine FGF receptor (FGFR) that is known, in other systems, to inhibit FGF binding to its receptor. When analyzed by SDS-PAGE and stained with Coomassie blue the 66 kDa fraction appeared as a single component. However, in some experiments it appeared more heterogeneous when stained with silver indicating the presence of minor components that may be a procedural artifact or isoforms of the same glycoprotein. The 66 kDa protein(s) migrated in isoelectric focusing with a pI of 7.4. The results are discussed in terms of the possible role of the 66 kDa glycoprotein as a protein involved in peptide-mediated cell signalling.

Bacterial binding to extracellular matrix proteins -- in vitro adhesion.

A binding assay was developed and used to study the binding of oral streptococcus to immobilized human fibronectin, laminin, vitronectin, fibrinogen, heparin, and collagen IV. The protein binding was dependent on the broth used for bacterial growth. The binding after growth in brain heart infusion broth, trypticase soy broth, Todd-Hewitt broth, and Dulbecco's modified Eagle's medium was examined. Most of the strains were able to bind to immobilized fibronectin and laminin, and to a minor extent vitronectin. Binding was not observed on immobilized fibrinogen, collagen IV, or heparin. Measured surface hydrophobicity correlated well with the bacterial binding strength to the proteins. Streptococcal incubation with putative inhibitors indicates multiple binding mechanisms of a lectin-like and protein nature, possibly involving protein receptors.

Zonal variation in the distribution of an alpha 1-acid glycoprotein glycoform receptor in human adrenal cortex.

Using a histochemical technique with three different alpha 1-acid glycoprotein glycoform one glycoform specific receptor has been identified in human adrenal cortex. The receptor is associated to alpha 1-acid glycoprotein glycoform B and alpha 1-acid glycoprotein glycoform C. The glycoform specific receptor was located in the cytoplasm of glomerulosa and outer fasciculata cells. The intensity of the reaction product decreased in the fasciculata, and no staining was seen in inner fasciculata and reticularis. Inhibition with the simple sugars, mannose and GlcNAc confirmed a lectin-like reaction. The binding activity was dependent on the presence of calcium ions and not on thiol reagents. Thus the lectin-like receptor may belong to the C-type lectin family. Using an antibody to alpha 1-acid glycoprotein the presence of alpha 1-acid glycoprotein was observed in the same location as the glycoform specific receptor. The binding of alpha 1-acid glycoprotein glycoform B and alpha 1-acid glycoprotein glycoform C to the glycoform specific receptor is inhibited by the steroid hormones cortisone, aldosterone, estradiol and progesterone but not by testosterone. The pronounced changes in the distribution of AGP and its glycoform receptors during cell differentiation in the adrenal cortex suggest that AGP and its complementary lectins belong to the group of lectins which control differentiation and spatial position.

Use of Lectin-Affinity Electrophoresis for Quantification and Characterization of Glycoforms of α-1 Acid Glycoprotein.

Quantification and characterization of glycoproteins may be achieved by lectin-affinity electrophoresis. Here we show the identification and quantification of three naturally occurring glycoforms of human serum α-1 acid glycoprotein (AGP) or orosomucoid. The method described here is two-dimensional (2D); a combination of lectin-affinity electrophoresis in the first dimension, and quantitative immunoelectrophoresis with specific antibodies against the glycoprotein in the second dimension. This type of affinity electrophoresis is carried out in agarose gels and was first termed crossed immunoaffinoelectrophoresis because it is a variant of quantitative crossed immunoelectrophoresis (1). For general descriptions, applications, and techniques of crossed immunoelectrophoresis the reader is referred to other manuals (2,3).

Separation of monoclonal antibodies from cell-culture supernatants and ascites fluid using thiophilic agarose.

A one-step purification procedure will yield monoclonal antibodies from cell-culture supernatants and ascites fluids. The chromatographic adsorbent is thiophilic argose, i.e., beaded agarose gel coupled with ligands of thiophilic nature, often with a sulfone group and a sulfur atom. The chromatographic procedure is simply adsorption, wash, elution. The procedure is simple, efficient, and inexpensive.

Lectin-like receptor for alpha 1-acid glycoprotein in the epithelium of the rat prostate gland and seminal vesicles.

BACKGROUND: A receptor for alpha 1-acid glycoprotein glycoforms AGP-B and AGP-C in the epithelium of the rat prostate gland and seminal vesicles is described. METHODS: The interaction between AGP-glycoforms and their receptor is a lectin-like interaction confirmed by inhibition of the binding by mannose and N-Acetyl-D-glucosamine. RESULTS: In vitro the receptor was also inhibited by the steroid hormones cortisone, aldosterone, progesterone, and estradiol, but not by testosterone. A significant regional variation in the expression of AGP-lectin receptor and in the localization of AGP was seen in rat prostate and seminal vesicles. The localization of the AGP lectin receptor is compared to the localization of glycoprotein AGP, and small differences are found. CONCLUSIONS: It is proposed the AGP receptors in the prostate and seminal vesicles belong to a group of lectins in the control of differentiation and organ formation.

Lectin staining of renal cell tumours with special emphasis on oncocytomas.

There are conflicting results respecting the segmental tubular origin of renal oncocytomas, a type of tumour said to be highly differentiated and benign, though metastases have been described. The aim of this study was, by applying different lectins on renal cell carcinomas (n = 50) and oncocytomas (n = 12), to search for patterns which could indicate a specific segmental origin of oncocytomas and perhaps elucidate the differentiation of this tumour. The following lectins were applied: jacalin, peanut agglutinin (PNA), wheat germ agglutinin (WGA), phytohemagglutinin E (PHA-E), concanavalin A (Con A), and Dolichos biflorus agglutinin (DBA). The results show that oncocytomas are positive when jacalin, a Lectin that stains distal tubules and collecting tubules, is used, supporting the view that the tumour derives from distal or collecting tubules. The staining of the oncocytomas is generally polarized as in normal kidney tubules underlining that the tumour is highly differentiated. Two oncocytomas with aggressive behaviour showed areas with a diffuse pattern suggesting lower differentiation.

Two lectin-like receptors for alpha 1-acid glycoprotein in mouse testis.

Three glycoforms of alpha 1-acid glycoprotein (AGP) were biotinylated to examine their binding in mouse testis by light microscopy. The transition from one stage to another in the spermatogenic cycle is marked with an appearance of a receptor for the Concanavalin A (Con A) non-reactive glycoform AGP-A in the cytoplasm of spermatocytes, young spermatids and Sertoli cells. This receptor disappears in the late stages of the spermatids. The Con-A intermediately reactive and the Con-A reactive glycoforms, AGP-B and AGP-C, showed weak reaction in the cytoplasm of spermatocytes, spermatids and Sertoli cells and, at the last stages in the spermatogenic cycle, a very strong reaction in the late elongated spermatids and the apical extensions of Sertoli cells. The interactions are lectin-like as confirmed by inhibition with simple sugars. In addition, the bindings were inhibited by steroid hormones. AGP-A was inhibited by testosterone, oestradiol and progesterone, while AGP-B and AGP-C were inhibited by mannose, GlcNAc, cortisone, aldosterone, oestradiol and progesterone. The receptors and the corresponding AGP glycoforms may be adhesion molecules between Sertoli cells and the spermatogenic cells and may have a function as a regulatory factor for spermatozoa development.

Appearance and cellular distribution of lectin-like receptors for alpha 1-acid glycoprotein in the developing rat testis.

A histochemical avidin-biotin technique with three different alpha 1-acid glycoprotein glycoforms showed pronounced alterations in the cellular localization of two alpha 1-acid glycoprotein lectin-like receptors during cell differentiation in the developing rat testis. The binding of alpha 1-acid glycoprotein glycoforms to their receptors is inhibited by steroids. Testosterone, oestradiol and progesterone inhibited the binding of alpha 1-acid glycoprotein glycoform A to its receptor. Cortisone, aldosterone, oestradiol and progesterone inhibited the binding of alpha 1-acid glycoprotein glycoforms B and C to their receptor. A difference in the cellular content of alpha 1-acid glycoprotein glycoforms and alpha 1-acid glycoprotein receptors separates the spermatocytes and the early spermatids from the late spermatids. The difference in receptor composition implies a difference in the effect of different steroid hormones. The Leydig cells contained alpha 1-acid glycoprotein and lectin-like receptors for one of the glycoforms of alpha 1-acid glycoprotein from birth. The interaction between alpha 1-acid glycoprotein glycoforms and their receptors may modulate the actions of testosterone and other steroids in the testis.

Hydrophobic interactions between gliadin and proteins and celiac disease.

Gliadin-protein interaction and its relationship to the pathogenesis hypotheses of celiac disease was investigated. Wheat germ agglutinin was not immunodetected in gliadin preparations. Peptic-tryptic gliadin digest was used to study the gliadin-protein interactions by crossed immunoelectrophoresis and affinity blotting. Biotinylated gliadin digest interacted with IgG and bovine serum albumin but not with several glycoproteins. Since albumin and IgG light chains are not glycosylated, this interaction is not lectin-like, neither completely immunological because of recognition of the IgG Fc fraction. Immobilized and boiled IgG was not recognized by gliadin digest as a lectin. Gliadin digest fractions from T-gel chromatography reduced the fluorescence intensity of cis-parinaric acid bound to albumin. The gliadin-protein interaction is not lectin-like or completely immunological but hydrophobic. Hydrophobicity of gliadins may contribute to the pathogenic events that result in celiac disease.

Biotin carboxylases in mitochondria and the cytosol from skeletal and cardiac muscle as detected by avidin binding.

Biotin carboxylases in mammalian cells are regulatory enzymes in lipogenesis and gluconeogenesis. In this study, endogenous biotin in skeletal and cardiac muscle was detected using avidin conjugated with alkaline phosphatase and applied in high concentrations to muscle sections. The avidin binding was subsequently visualized by histochemical demonstration of the alkaline phosphatase activity. All cardiac muscle cells showed high affinity for avidin with only the nuclei and the intercalated discs remaining unstained. In skeletal muscle a diffuse reaction could be detected in the sarcoplasm of the muscle fibres. A granular reaction was noted in the same fibres that showed activity for succinic dehydrogenase. The specificity of the coloured reaction product in the muscle sections was investigated and is suggested to be caused by avidin binding to biotin moieties in mitochondria and the cytosol. Mitochondrial and cytosolic preparations of skeletal muscle were electrophoresed in sodium dodecyl sulphate gels. After blotting and incubation with conjugated avidin, two bands with molecular weights of 75 kDa and 130 kDa respectively were evident in the mitochondrial preparation. It is suggested that the 75-kDa band represents comigration of the biotin-containing subunits of propionyl-CoA carboxylase and methylcrotonyl-CoA carboxylase. The 130-kDa band may represent the biotin-containing pyruvate carboxylase. In the cytosolic preparation a 270-kDa band was stained in blots that had been incubated with conjugated avidin; this band is suggested to represent acetyl-CoA carboxylase. A 190-kDa cytosolic band might be a cleavage product of acetyl-CoA carboxylase. We propose that using alkaline phosphatase-conjugated avidin it is possible to detect the mitochondrial and cytosolic biotin-dependent carboxylases in striated muscle.

Affinity methods with lectins: a tool to identify canine alkaline phosphatase isoenzymes.

Affinity methods were used to characterize selective interactions of alkaline phosphatase (ALP) isoenzymes from different dog tissues with lectins. Specific lectins were used to identify liver, intestinal and steroid-induced ALP isoenzymes in serum from dogs with Cushing syndrome or steroid-treated dogs. For the first approach, 12 lectins were assayed by affinity dots. Selective interactions were found among wheat germ agglutinin (WGA), jacalin, con A (concanavalin A) and Helix pomatia agglutinin (HPA) and several ALP-containing samples. These four reactive lectins were assayed by line electrophoresis with lectins in holes. A strong reactivity of con A with all isoenzymes was found, although the patterns were different. WGA interacted with intestinal, bone marrow extracts and Cushing syndrome serum. Jacalin changed the electrophoretic patterns of intestinal and liver ALP, and Cushing serum. Finally, by crossed electrophoresis with lectins in gels, it was possible to distinguish among hepatic or intestinal ALPs and the steroid-induced isoenzyme in serum. Affinity electrophoresis with lectins provided a clear separation and identification of the different dog ALP isoenzymes.

Fucose expression in skeletal muscle: a lectin histochemical study.

Binding sites for three fucose specific lectins, Aleuria aurantia agglutinin (AAA), Lotus tetragonolobus agglutinin (LTA) and Ulex europeus I agglutinin (UEA I), were investigated in sections from normal human and rat muscles, in muscle from patients with Duchenne muscular dystrophy (DMD) and in denervated and devascularized rat muscle. In normal human and rat muscle AAA detected fucosylated glycocompounds in the sarcoplasm, sarcolemma, interfibre connective tissue and vascular structures. In normal human muscle addition of fucose to the AAA incubation medium or treatment of the sections with formaldehyde followed by periodic oxidation before lectin incubation strongly inhibited the staining at all sites other than endothelial cells. In normal rat muscle the same staining procedures strongly inhibited the AAA binding at all sites other than the sarcolemma. Incubation with LTA resulted in a diffuse reaction around the vascular structures in rat muscle, while in human muscle a moderate, homogeneous staining was present in all muscle fibres. Treatment of the sections with formaldehyde and periodic acid before incubation with LTA resulted in strongly labelled muscle capillaries in both human and rat muscle. The only elements in the muscle tissues that were stained with UEA I were human endothelial cells. In denervated and devascularized rat muscle incubation with AAA revealed a novel fucose expression that appeared intracellularly in some necrotic fibres. The AAA-positive fucose residues in the sarcolemma of normal muscle fibres that were resistant to periodic acid oxidation could not be shown by AAA in denervated muscle. In DMD muscle a cryptic sarcolemmal fucose expression could be shown with AAA.(ABSTRACT TRUNCATED AT 250 WORDS)

Concanavalin A affinity immunoelectrophoresis of slowly migrating glycoproteins by chemical charge modification.

A method for the study of slowly migrating glycoproteins by lectin-affinity immunoelectrophoresis is described. The principle of the method is to modify chemically the polypeptide part of the glycoprotein in question to increase the net negative charge of the molecule without affecting the carbohydrate parts. In a model experiment, desialylated human alpha 1-acid glycoprotein is modified and it is shown by lectin affinity immunoelectrophoresis that glycan-bound sialic acid does not influence the binding of human alpha 1-acid glycoprotein to concanavalin A. Additionally, the method is used to study the carbohydrate-based microheterogeneity of slowly migrating mouse serum glycoproteins, and hitherto undetected microheterogeneity inherent in mouse transferrin and mouse haemopexin is detected and described in normal and inflammatory mice.

The silver staining procedure of sodium dodecyl sulfate-gels may be accelerated by shortening fixation time.

In most silver staining methods the first step in the staining of proteins separated after sodium dodecyl sulfate-polyacrylamide gel electrophoresis is a rather protracted fixation of the gels. Optimum fixation should be short, cause no background staining and effectively immobilize the proteins in the gel without masking the proteins for reaction with the staining solution. Further, the concentration of the fixing compounds should be as low as possible due to the potential toxicity of fixatives. Fixation for only 5 min with mixtures of very low concentrations of formaldehyde and glutaraldehyde in ethanol, or a solution of formaldehyde or glutaraldehyde in picric acid and ethanol, fulfill these demands, provided that the gels were prefixed in ethanol-acetic acid for 10 min. As a consequence of these results a fast and sensitive silver staining procedure is proposed.

Mosaic lectin and enzyme staining patterns in rat skeletal muscle.

We compared the localizations of lectin binding and activity for myosin ATPase and succinic dehydrogenase in sections of the gracilis, soleus, and masseter muscles from 10- and 60-day-old rats. In the 60-day-old rats, incubation of the muscle sections with the lectins ConA, GS-II, HPA, and jacalin gave rise to a mosaic staining pattern, especially in the gracilis muscle, in which the same fibers were strongly stained for ConA, GS-II, and HPA, whereas the staining with jacalin in these fibers was weak, and vice versa. There was no correspondence in the staining patterns for the enzymes and the lectins. In the masseter muscle only GS-II gave rise to distinct differences in the staining intensity between muscle fibers. In 10-day-old rats all fibers in the muscles were moderately stained with ConA, HPA, and jacalin, whereas a chessboard staining pattern could be observed after incubation with GS-II. In an extract of hindleg muscle from 60-day-old rats there was strong affinity for ConA and HPA and weak affinity for GS-II and jacalin, as shown by dot-blotting. After electrophoresis and blotting to nitrocellulose membranes, three muscle protein bands with apparent molecular weights of 100,000, 90,000, and 43,000 showed affinity for ConA, HPA, and GS-II, whereas no bands were jacalin positive. The complex lectin staining pattern in skeletal muscle might be related to development, specialization, and function of the muscles.