Showing papers by "Ajit Varki published in 1995"

Differential colon cancer cell adhesion to E-, P-, and L-selectin: role of mucin-type glycoproteins.

Abstract: E-, P-, and L-selectin support the adhesion of leukocytes to the vessel wall through the recognition of specific carbohydrate ligands, which often contain sialylated, fucosylated lactosamines such as sialyl Lewis x [sLex; Neu5Ac alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc-]. E-selectin expressed by activated endothelium has been shown to support the adhesion of sLex-bearing colon cancer cells. In the present study, we examine the interactions of multiple colon cancer cell lines with all three selectins. Three colon cancer cell lines (LS 180, T84, and COLO 205) bound to recombinant purified E-, P-, and L-selectin. The colon cancer line COLO 320 bound to P- and L-selectin but not E-selectin; conversely, HT-29 cells bound E-selectin but not P- and L-selectin. Caco-2 showed little or no interaction with any of the three selectins. Treatment of the cells with O-sialoglycoprotease from Pasteurella haemolytica, an enzyme that selectively cleaves mucin-type O-linked glycoproteins, reduced binding to purified P- and L-selectin in all cases. In addition, recombinant soluble P- and L-selectin bound to affinity-purified mucins from all adherent tumor cell lines. Of the four tumor cell lines that interacted with E-selectin, O-glycoprotease treatment substantially diminished adhesion of LS 180 and T84, had little effect on COLO 205, and failed to inhibit the binding of HT-29. As predicted by these data, E-selectin showed substantial binding only to mucins purified from LS 180 and T84. These findings suggest that L- and P-selectin interact primarily with mucin-type ligands on colon cancers, whereas E-selectin can recognize both mucin and nonmucin ligands. Binding of the colon cancer lines to purified selectins correlates with their adhesion to activated endothelial cells (E-selectin-dependent), platelets (P-selectin-dependent), and neutrophils (L-selectin-dependent). These differential tumor cell-selectin interactions may influence metastatic spread and may also contribute to the observed variability in host response to tumor progression.

Co-localization of hydrolytic enzymes with widely disparate pH optima: implications for the regulation of lysosomal pH

Abstract: Lysosomes are traditionally defined by their acidic interior, their content of degradative ‘acid hydrolases’, and the presence of distinctive membrane proteins. Terminal degradation of the N-linked oligosaccharides of glycoproteins takes place in lysosomes, and involves several hydrolases, many of which are known to have acidic pH optima. However, a sialic acid-specific 9-O-acetyl-esterase and a glycosyl-N-asparaginase, which degrade the outer- and inner-most linkages of N-linked oligosaccharides, respectively, both have pH optima in the neutral to alkaline range. By immunoelectron microscopy, these enzymes co-localize in lysosomes with several conventional acid hydrolases and with lysosomal membrane glycoproteins. Factors modifying the pH/activity profiles of these enzymes could not be found in lysosomal extracts. Thus, the function of the enzymes with neutral pH optima must depend either upon their minimal residual activity at acidic pH, or upon the possibility that lysosomes are not always strongly acidic. Indeed, when lysosomes are marked in living cells by uptake of fluorescently labeled mannose 6-phosphorylated proteins, the labeled organelles do not all rapidly accumulate Acridine Orange, a vital stain that is specific for acidic compartments. One plausible explanation is that lysosomal pH fluctuates, allowing hydrolytic enzymes with a wide range of pH optima to efficiently degrade macromolecules.

Structure of the N-linked oligosaccharides of MHC class I molecules from cells deficient in the antigenic peptide transporter. Implications for the site of peptide association.

Abstract: Class I molecules are N-linked glycoproteins encoded by the MHC. They carry cytosolic protein-derived peptides to the cell surface, displaying them to enable immune surveillance of cellular processes. Peptides are delivered to class I molecules by the transporter associated with Ag processing (TAP). Peptide association is known to occur before exposure of class I molecules to the medial Golgi-processing enzyme alpha-mannosidase II, but there is limited information regarding the location or timing of peptide binding within the earlier regions of the endoplasmic reticulum (ER)-Golgi pathway. A reported association of newly synthesized class I molecules with the ER chaperonin calnexin raises the possibility of persistence of the monoglycosylated N-linked oligosaccharide (NLO) Glc1Man8GlcNAc2, known to be recognized by this lectin. To explore these matters, we determined the structure of the NLOs on the subset of newly synthesized class I molecules awaiting the loading of peptide. We pulse-labeled murine MHC H-2Db class I molecules in RMA/S cells, which lack one of the TAP subunits, causing the great majority of the molecules to be retained for prolonged periods in an early secretory compartment, awaiting peptide binding. MHC molecules pulse-labeled with [3H]glucosamine were isolated, the NLOs specifically released and structurally analyzed by a variety of techniques. Within the chosen window of biosynthetic time, most Db molecules from parental RMA cells carried mature NLOs of the biantennary complex-type, with one to two sialic acid residues. In RMA/S cells, such chains were in the minority, the majority consisting of the precursor forms Man8GlcNAc2 and Man9GlcNAc2. No glucosylated forms were detected, nor were the later processing intermediates Man5-7GlcNAc2 or GlcNAc1Man4-5GlcNAc2. Thus, most Db molecules in TAP-deficient cells are retained in an early compartment of the secretory pathway, before the point of first access to the Golgi alpha-mannosidase I, which trims alpha 1-2 linked mannose residues, but beyond the point where the alpha 1-3-linked glucose residue is finally removed by the ER glucosidase II. Thus, structural analysis of NLOs on class I molecules within a defined biosynthetic window has established a biochemical measure of the timing of peptide association.

Analysis of Monosaccharides

Abstract: This unit presents methods for assaying sialic acids, reducing sugars, and hexosamines. The BCA assay detects free reducing terminii in sugars released from glycoconjugates by appropriate treatments. Assays employing Ehrlich reagent (DMAB) detect hexosamines and N-acetylhexosamines, including a method for hydrolyzing the glycosidic linkages of the hexosamines and a method for re-N-acetylation. The TBA and DMB assays can be used to quantitate and fractionate free forms of many types of sialic acids. Techniques for liberating the sialic acids from the parent glycoconjugates are also provided.

Analysis of oligosaccharide negative charge by anion-exchange chromatography.

Abstract: This unit presents the analysis of negative charge on labeled N- or O-linked oligosaccharides. These protocols may be used in the initial screening of oligosaccharides to detect negative charge, for analytical or preparative separation of oligosaccharides based on their negative charge, or to analyze the type of negative charge found on the oligos accharides. The Basic Protocol describes the use of anion-exchange (QAE-Sephadex) chromatography with stepwise elution for estimating the number of negative charges on an oligosaccharide sample derived from glycosidase treatment of a glycoprotein. In an Alternate Protocol, gradient elution is used for the preparative separation of oligosaccharides based on negative charge. A Support Protocol describes a method for measuring loss of or change in negative charge after treatment of the oligosaccharide sample with mild acid and/or phosphatases.

Fractionation and Analysis of Neutral Oligosaccharides by HPLC

Abstract: This unit describes the fractionation and analysis of neutral oligosaccharides by high-performance liquid chromatography (HPLC) on bonded amine columns. Separation is based upon hydrogen bonding between the NH2 groups of the column and the hydroxyl groups of the oligosaccharides. A support protocol describes the reduction and desalting of neutral oligosaccharides with sodium borohydride.