title: Determinants in the N-terminal domains of galectin-3 for secretion by a novel pathway circumventing the endoplasmic reticulum-Golgi complex.
abstract: Galectin-3 is a beta-galactoside-binding protein that is secreted from many cells although the protein lacks a signal sequence for transfer into the endoplasmic reticulum and Golgi compartments and entry into classical secretory pathways. Previously it was shown that attachment of the first 120 amino acid residues of the N-terminal sequence of hamster galectin-3 to the cytoplasmic protein chloramphenicol acetyltransferase (CAT) supported the rapid secretion of the fusion protein from transiently transfected Cos cells under conditions in which CAT protein was not secreted. Here we report that progressive N-terminal truncation gradually reduced secretion of the fusion proteins, eventually to very low levels compared with the starting product, but did not totally eliminate secretion until a significant majority of the sequence was removed. Mutant CAT fusion proteins containing internal deletions in residues 97-120 of the galectin-3 N-terminal sequence were also secreted to a similar extent to the starting product, but further deletion of residues 89-96 abolished detectable secretion. Proline to alanine mutagenesis of the sequence YP(90)SAP(93)GAY in two secretion-competent CAT fusion proteins greatly reduced or abolished their secretion, whereas similar mutagenesis of proline pairings present elsewhere in the galectin-3 N-terminal segments of these proteins had no effect. The results indicate that this sequence is one essential determinant for secretion of galectin-3-CAT fusion proteins and by inference galectin-3, at least from transfected Cos cells. However, the short sequence of residues 89-96 by itself is insufficient to direct secretion of CAT fusion proteins and appears to be active only in the context of a larger portion of the galectin-3 N-terminal sequence.
authors.affiliation: National Institute for Medical Research, Mill Hill, London, UK.
authors.forename: R P
authors.initials: RP
authors.lastname: Menon
authors.forename: R C
authors.initials: RC
authors.lastname: Hughes
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000943:Antigens, Differentiation / Q000235:genetics* / Q000378:metabolism; D015153:Blotting, Western; D019556:COS Cells; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D006224:Cricetinae; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D037502:Galectin 3; D006056:Golgi Apparatus / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000235:genetics; D017384:Sequence Deletion; D013997:Time Factors; D014162:Transfection
chemical_list: D000943:Antigens, Differentiation; D037502:Galectin 3; D011993:Recombinant Fusion Proteins; D015500:Chloramphenicol O-Acetyltransferase