Protein core-dependent glycosaminoglycan modification and glycosaminoglycan-dependent polarized sorting in epithelial Madin-Darby canine kidney cells.
The proteoglycan serglycin (SG) fused to green fluorescent protein (GFP) is secreted predominantly from the apical surface of polarized epithelial Madin-Darby canine kidney (MDCK) cell monolayers, but the minor fraction secreted basolaterally carries more intensely sulfated glycosaminoglycan (GAG) chains (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). To investigate whether the domain with GAG attachment sites in SG (i) is sufficient to drive apical protein sorting and (ii) independently generates the sulfation differences observed in the apical and basolateral pathways, the GAG domain of SG was fused into the junction of rat growth hormone (rGH) and GFP and expressed in MDCK cells, either with or without two N-glycosylation sites in the rGH part. Both variants acquired chondroitin sulfate GAG chains and were secreted predominantly to the apical medium, to the same extent as rGH-GFP with two N-glycosylation sites only, and different from the nonsorted variant lacking glycosylation sites. Transfer of the GAG attachment domain from SG to the new rGH context abolished the differences in sulfation intensity and positions observed for SG in the apical and basolateral secretory routes. Thus, these differences are coded by elements outside the GAG attachment domain.
Department of Molecular Biosciences, University of Oslo, Box 1041, Blindern, 0316 Oslo, Norway.
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D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002809:Chondroitin Sulfates / Q000378:metabolism; D004187:Disaccharides / Q000378:metabolism; D004285:Dogs; D004847:Epithelial Cells / Q000378:metabolism*; D006025:Glycosaminoglycans / Q000737:chemistry / Q000378:metabolism*; D006031:Glycosylation; D049452:Green Fluorescent Proteins / Q000378:metabolism; D013006:Growth Hormone / Q000378:metabolism; D008969:Molecular Sequence Data; D015202:Protein Engineering; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011509:Proteoglycans / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D033921:Vesicular Transport Proteins / Q000378:metabolism
D004187:Disaccharides; D006025:Glycosaminoglycans; D011509:Proteoglycans; D011993:Recombinant Fusion Proteins; D033921:Vesicular Transport Proteins; C075430:serglycin; D049452:Green Fluorescent Proteins; D013006:Growth Hormone; D002809:Chondroitin Sulfates