Increased LAMP-2 polylactosamine glycosylation is associated with its slower Golgi transit during establishment of a polarized MDCK epithelial monolayer.
An endogenous Madin-Darby canine kidney (MDCK) lysosomal membrane glycoprotein that exhibits a basolateral targeting pathway to the lysosome is shown here to exhibit significant N-terminal amino acid sequence identity to lysosomal associated membrane proteins (LAMP-2) of other species. During establishment of the MDCK monolayer after only 1 d of culture, this canine LAMP-2 has a larger molecular size (110 kDa) than following formation of a confluent monolayer after 3 d of culture (100 kDa) due to the increased presence of N-linked polylactosamine oligosaccharide chains. Neither polylactosamine glycosylation of LAMP-2 in MDCK cells nor truncation of N-linked oligosaccharide chains of LAMP-2 in a ricin-resistant MDCK-RCAR cell line influenced the basolateral polarity of its targeting. However, the rate of basolateral delivery of LAMP-2 in MDCK cells plated for 3 d was significantly faster (t1/2 = 28 min) than in 1-d cells (t1/2 = 40 min); in MDCK-RCAR cells the rate of basolateral delivery at both 1 and 3 d of plating was similar (t1/2 = 40 min). The rate differential in MDCK cells occurred after arrival of LAMP-2 to the Golgi apparatus because the rate of acquisition of endoglycosidase H resistance was the same (t1/2 = 25 min) at both days of plating. The rate of transit of LAMP-2 through the Golgi apparatus to the basolateral domain was therefore far more rapid (approximately 4-fold) in 3 d compared with 1-d MDCK cultures. The increased polylactosamine glycosylation of MDCK LAMP-2 at early times of plating during the establishment of a confluent epithelial monolayer may thus be related to its longer residence time in the Golgi apparatus.
Département de pathologie, Université de Montréal, Québec, Canada.
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D000595:Amino Acid Sequence; D000606:Amino Sugars / Q000378:metabolism*; D000818:Animals; D015703:Antigens, CD; D002460:Cell Line; D016764:Cell Polarity; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D004847:Epithelial Cells; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism*; D007668:Kidney; D037102:Lectins / Q000378:metabolism; D051907:Lysosomal Membrane Proteins; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011134:Polysaccharides / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D013997:Time Factors
D000606:Amino Sugars; D015703:Antigens, CD; D037102:Lectins; D051907:Lysosomal Membrane Proteins; D008562:Membrane Glycoproteins; D011134:Polysaccharides; C066929:polylactosamine