N-Glycan synthesis in the apical and basolateral secretory pathway of epithelial MDCK cells and the influence of a glycosaminoglycan domain

Abstract

Different classes of glycans are implicated as mediators of apical protein sorting in the secretory pathway of epithelial cells, but recent research indicates that sorting to the apical and basolateral surfaces may occur before completion of glycan synthesis. We have previously shown that a proteoglycan (PG) core protein can obtain different glycosaminoglycan (GAG) structures in the apical and basolateral secretory routes (Tveit H, Dick G, Skibeli V, Prydz K. 2005. A proteoglycan undergoes different modifications en route to the apical and basolateral surfaces of Madin-Darby canine kidney cells. J Biol Chem. 280:29596-29603) of epithelial Madin-Darby canine kidney (MDCK) cells. We have now also determined the detailed N-glycan structures acquired by a single glycoprotein species in the same apical and basolateral secretory pathways. For this purpose, rat growth hormone (rGH) with two N-glycan sites (rGH-2N) inserted into the rGH portion (NAS and NFT) was fused to green fluorescent protein (GFP) and expressed in MDCK cells. Immunoisolated rGH variants were analyzed for site occupancy and N-glycan structure by mass spectrometry. The extent of NAS and NFT site occupancy was different, but comparable for rGH-2N secreted apically and basolaterally. Microheterogeneity existed for the glycans attached to each N-glycan site, but no major differences were observed in the apical and basolateral pathways. Transfer of the GAG modification domain from the PG serglycin to the fusion site of rGH-2N and GFP allowed polymerization of GAG chains onto the novel protein variant and influenced the microheterogeneity of the N-glycans toward more acidic glycans, but did not alter the relative site occupancy. In conclusion, no major differences were observed for N-glycan structures obtained by the expressed model proteins in the apical and basolateral secretory pathways of epithelial MDCK cells, but insertion of a GAG attachment domain shifted the N-glycans to more acidic structures.

The Epithelial Calcium Channel TRPV5 Is Regulated Differentially by Klotho and Sialidase

“…Previous studies have shown that intracellular N-glycans contribute to signaling processes, dimer formation, membrane trafficking and polarized sorting of glycoproteins to the apical surface of epithelial cells (8,9). Extracellular N-glycans of plasma membrane glycoproteins are involved in signaling processes, apoptosis and cell-cell contacts.…”

Evaluation of Multivalent Sialylated Polyglycerols for Resistance Induction in and Broad Antiviral Activity against Influenza A Viruses

“…All viral mutants bound strongly to all six aminoglycosides presented on the array, whereas WT virus recognized sisomicin sulfate, neomycin sulfate and, with weak binding intensity, also gentamicin sulfate and kanamycin sulfate. Taken together, these data indicate that the mutant viruses lost the high affinity binding to the typical IAV glycan receptors, while maintaining or acquiring the ability to bind different glycan receptors of the vast variety expressed on MDCK cells. − …”

“…The reduced affinity for the prototypical 6′-Neu5Ac receptor could lead the mutant viruses to bind these alternative receptors. It is entirely possible that we did not detect additional receptors of the mutant viruses here because the arrays display a limited fraction of short glycans present on tissue culture cells − or in respiratory tissues …”

“…Despite a reduction of affinity for the receptors used by WT virus, the mutant viruses displayed growth curves similar to WT virus (Figure A). There is a broad variety of glycans present in the lung and also on MDCK II cells. − Hence, changes in receptor specificity or affinity do not necessarily have a negative impact on viral fitness or infectivity, as the IAV can apparently switch to alternatively preferred determinants . It appears that LPG 10 SA 0.70 and dPG 10 SA 0.15 are too similar to LPG 10 SA 0.40 and the difference in SA density or flexibility is not sufficient to overcome the altered receptor preference of the mutant viruses, resulting in cross-resistance (Figure B).…”

Separation optimization of long porous‐layer open‐tubular columns for nano‐LC–MS of limited proteomic samples

“…The in‐gel‐digested rat liver sample was prepared according to a previously described procedure . In brief, 15 μL (40 μg/μL) of rat liver extract was loaded onto the gel.…”