Identification and characterization of endogenous galectins expressed in Madin Darby canine kidney cells.
Madin Darby canine kidney (MDCK) cells are a well characterized epithelial cell line used to study mechanisms of polarized delivery. As glycans on apically expressed proteins have been identified as targeting signals, and crosslinking by the abundant galectin-3 has been implicated in the mechanism of glycan-dependent sorting, we wanted to identify other members of the galectin (Gal) family expressed in MDCK cells. By analyzing intron-exon boundaries, we identified canine genes that were highly homologous to mammalian Gal-1, 2, 3, 4, 7, 8, 9, and 12, and galectin-related HSPC159 and GRIFIN. Transcripts for Gal-2 and -12 were not detected in MDCK cells, but we found transcript levels for Gal-3 > Gal-9 > Gal-8 > Gal-1 ⋙ Gal-4 > Gal-7. Canine Gal-1, -2, -3, -4, -7, -8, -9, and -12 were cloned and expressed in Escherichia coli as GST fusion proteins to characterize binding specificities on arrays of synthetic glycans on glass slides from Core H of the NIH Consortium for Functional Glycomics. Individual expression of the N-terminal (GST-Gal-9N) and C-terminal (GST-Gal-9C) carbohydrate recognition domains greatly improved protein yield and the ability to characterize Gal-9 binding on the array. Canine galectins differentially bound sulfated disaccharides as well as human blood groups A, B, and H on both N-glycans and linear glycan structures on the array. Analysis of GST-Gal-1, -3, -4, -7, -8, -9N, and -9C binding to immunopurified human MUC1 expressed in MDCK cells revealed a preference for binding GST-Gal-3 and -9, which interestingly reflects the two most abundant galectins expressed in MDCK cells.
Department of Medicine, Renal-Electrolyte Division, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA.
Paul A
PA
Poland
Christine
C
Rondanino
Carol L
CL
Kinlough
Jamie
J
Heimburg-Molinaro
Connie M
CM
Arthur
Sean R
SR
Stowell
Dave F
DF
Smith
Rebecca P
RP
Hughey
D000818:Animals; D002460:Cell Line; D004285:Dogs; D037161:Galectins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D037161:Galectins; D011994:Recombinant Proteins