Determinants in the N-terminal domains of galectin-3 for secretion by a novel pathway circumventing the endoplasmic reticulum-Golgi complex

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 e ndoplasmic reticulum and Golgi compartments and entry into classical secret ory pathways. Previously it was shown that attachment of the first 120 amin o acid residues of the N-terminal sequence of hamster galectin-3 to the cyt oplasmic protein chloramphenicol acetyltransferase (CAT) supported the rapi d secretion of the fusion protein from transiently transfected Cos cells un der conditions in which CAT protein was not secreted. Here we report that p rogressive 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 th e sequence was removed. Mutant CAT fusion proteins containing internal dele tions in residues 97-120 of the galectin-3 N-terminal sequence were also se creted to a similar extent to the starting product, but further deletion of residues 89-96 abolished detectable secretion. Proline to alanine mutagene sis of the sequence YP(90)SAP(93)GAY in two secretion-competent CAT fusion proteins greatly reduced or abolished their secretion, whereas similar muta genesis of proline pairings present elsewhere in the galectin-3 N-terminal segments of these proteins had no effect. The results indicate that this se quence 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 th e context of a larger portion of the galectin-3 N-terminal sequence.