Apoptotic cells selectively uptake minor glycoforms of vitronectin from serum

Abstract

Apoptosis profoundly alters the carbohydrate layer coating the membrane of eukaryotic cells. Previously we showed that apoptotic cells became reactive with the α2,6-sialyl-specific lectin from Sambucus nigra agglutinin (SNA), regardless of their histological origin and the nature of the apoptotic stimulus. Here we reveal the basis of the phenomenon by showing that in apoptotic cancer cell lines SNA reactivity was mainly associated with a 67 kDa glycoprotein which we identified by MALDI-TOF/TOF and immunoblot analysis as bovine vitronectin (bVN). bVN was neither present in non-apoptotic cells, nor in cells induced to apoptosis in serum-free medium, indicating that its uptake from the cell culture serum occurred only during apoptosis. The bVN molecules associated with apoptotic cancer cell lines represented minor isoforms, lacking the carboxyterminal sequence and paradoxically containing a few α2,6-linked sialic acid residues. Despite their poor α2,6-sialylation, these bVN molecules were sufficient to turn apoptotic cells to SNA reactivity, which is a late apoptotic event occurring in cells positive to both annexin-V and propidium iodide. Unlike in cancer cell lines, the major bVN form taken up by apoptotic neutrophils and mononuclear cells was a 80 kDa form. In apoptotic SW948 cells we also detected the α2,6-sialylated forms of the stress-70 mitochondrial precursor (mortalin) and of tubulin-β2C. These data indicate that the acquisition of vitronectin isoforms from the environment is a general, although cell specific phenomenon, potentially playing an important role in post-apoptotic events and that the α2,6-sialylation of intracellular proteins is a new kind of posttranslational modification associated with apoptosis.

Identification of novel plasma glycosylation-associated markers of aging

“…The following lectins (Sigma) were labelled with digoxigenin (Roche) as indicated by the provider and used for the detection of the indicated sugar chains: Sambucus nigra (SNA) for α2,6-linked sialic acid [ 32 ]; Erythrina cristagalli (ECL) for terminal galactose [ 33 ]; Griffonia simplicifolia II (GSA-II), for terminal N-acetylglucosamine (GlcNAc) [ 34 ]; erythroagglutinin from Phaseolus vulgaris , for bisecting GlcNAc [ 35 ] and Ulex europeus , for L-fucose [ 36 ]. Blotting and detection were performed as previously described [ 76 ].…”

Vitronectin—Master controller or micromanager?

“…The importance of such interactions is illustrated by the appearance of disease associated with aberrant post‐translational GAG‐modifications. Aberrant glycation of Vn is linked with impaired endothelial cell function associated with hyperglycaemia, vascular disease and diabetes A truncated hypo‐sialylated form of VN has been recently associated with cells in late‐stage apoptosis , suggesting specific isoforms of VN may support the removal of apoptotic bodies. Interestingly, denatured glycosylated VN inhibits the apoptosis of neutrophils in inflammed tissues .…”

Black Elder and Its Constituents: Molecular Mechanisms of Action Associated with Female Reproduction

“…There is evidence that the positive action of elderberry on vascular health is due to the presence of anthocyanins, especially cyanidin-3-O-glucoside [59,62]. Moreover, elderberry agglutinins can selectively bind to apoptotic cells, and can be involved in the execution of mitochondrial apoptosis in various cell types [81]. Finally, elderberry agglutinin can block vascular endothelial growth factor (VEGF)-induced angiogenesis [82], which plays an important role in tumor growth.…”