Evidence for glycoconjugate in nociceptive primary sensory neurons and its origin from the Golgi complex.

Isolation and characterization of gastric microsomal glycoproteins. evidence for a glycosylated β-subunit of the H+/K−-ATPase

High resolution immunolabeling applying the protein A-gold technique and carbohydrate cytochemistry using lectin-gold labeling on Lowicryl K4M and thawed-frozen thin sections are most useful approaches for the detection of protein antigens and lectin binding sites in intracellular organelles and the plasma membrane. They provided the basis for modern electron microscopic studies on protein glycosylation reactions and the identification of their subcellular localization as reviewed here. These studies have demonstrated organelle subcompartments and the cell type-specific compartmentation of endoplasmic reticulum and Golgi apparatus-associated glycosylation reactions. The other subject reviewed in this paper is cell surface glycoconjugates, as they are expressed in relation to specific cell types present in various organs and during cellular differentiation processes.

Protein glycosylation in the endoplasmic reticulum and the Golgi apparatus and cell type-specificity of cell surface glycoconjugate expression: analysis by the protein A-gold and lectin-gold techniques.

We report the use of a novel hapten system for lectin cytochemistry. Various lectins conjugated to the steroid hapten digoxigenin (DIG) and monospecific anti-digoxigenin antibodies were applied for the light and electron microscopic detection of glycoconjugates in tissue sections. Both IgG and Fab' anti-DIG antibodies were complexed to particles of colloidal gold and compared to commercially available alkaline phosphatase and horseradish peroxidase conjugated Fab' as general second step reagents. The three different markers performed equally well on paraffin sections whereas the gold-labeled antibodies were superior reagents for semithin and ultrathin sections of Lowicryl K4M embedded tissues. In conjunction with the latter marker, no pretreatment to abolish endogenous enzyme activity was necessary. At the light microscope level, gold signal amplification by the photochemical silver reaction was required. DIG, in contrast to biotin, does not occur in animal tissues thus eliminating the need for blocking reactions prior to lectin incubation. Compared to affinity techniques using glycoprotein-gold complexes as second step reagent the DIG hapten system required smaller amounts of lectins. The staining patterns were indistinguishable from those obtained in other lectin-gold techniques and the specificity of the labeling could be demonstrated in sugar inhibition tests.

Lectin — digoxigenin conjugates: a new hapten system for glycoconjugate cytochemistry

Lectins are proteins and glycoproteins extracted predominantly from plants which have the capacity to bind sugars specifically. This property makes them of interest for histopathology since they will bind to saccharides forming parts of glycoproteins and glycolipids of tissue constituents. Lectins have and can be used as reagents for mucin histochemistry, to identify specific cells, in the recognition of glycoprotein alterations in disease states, in studies of infectious diseases, and in the assessment of glycoconjugate alterations occurring with malignancy. They can be used for both light microscopic and ultrastructural localisation and various methods are available. It is important though, to consider the nature of the glycoconjugates under study and select lectins appropriately because of their varying specificities and binding characteristics. A panel of lectins should be used to study a particular configuration. Care should be taken with tissue fixation and processing. It must be remembered that an open and critical mind should be kept concerning interpretation of results. At the present time lectins have a limited value diagnostically, but the binding of Ulex europeus agglutinin to endothelium is certainly of value.

The Use of Lectins in Histopathology

A conjugate of peanut lectin with horseradish peroxidase (PL-HRP) has been employed for ultrastructural localization of glycoprotein with presumed terminal galactose residues in mouse alimentary epithelial cells. The PL-HRP conjugate imparted electron opacity in sites that stain at the light microscopic level, as for example, Golgi cisternae in surface epithelial cells of the stomach and in superficial and deep crypt cells and goblet cells of the large intestine. Ultrastructural staining revealed that Golgi cisternae intermediate between the trans and cis faces stained selectively in these sites. Secretion stored in secretory granules or Golgi vesicles in the cells lacked affinity for PL-HRP conjugate, however. Selective staining of intermediate Golgi cisternae in cells with unreactive secretory product is interpreted as indicating the site of galactosyl transferase activity and a location where galactose occurs transitorily as the terminal sugar in the glycoprotein side chains. The luminal aspect of the surface epithelial cells in the stomach and columnar cells in the colon also stained, but with some variability. Staining of these surfaces was considered possibly attributable to PL affinity of some of the secretory glycoprotein which, after absorbing to the cell surface, lost terminal sialic acid through action of luminal enzyme. PL-HRP conjugate stained granules in pancreatic zymogen cells near the block surface but not in other cells, presumably because of limited penetration of reagent. Secretion on the surface of pancreatic acinar cells or in the lumen also exhibited affinity for PL-HRP complex as did the luminal surface of gastric chief cells. Staining of secretion in the pancreatic zymogen cells and gastric chief cells for galactose appeared inconsistent with lack of evidence for presence of glycoprotein in these sites which failed to stain with the periodic acid-Schiff or periodic acid-thiocarbohydrazide-silver proteinate methods for demonstrating glycoprotein at the light and electron microscopic levels. This discrepancy points to possible selective binding of PL-HRP conjugate to a moiety other than terminal galactose of glycoprotein in a few histologic sites. These results demonstrate the applicability of the PL-HRP technique at the ultrastructural level and provide information concerning the chemical structure of epithelial cell glycoproteins and their biosynthesis.

Ultrastructural visualization of galactosyl residues in various alimentary epithelial cells with the peanut lectin-horseradish peroxidase procedure

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Ultrastructural visualization of galactosyl residues in various alimentary epithelial cells with the peanut lectin-horseradish peroxidase procedure