Menon R P, Hughes R C
National Institute for Medical Research, Mill Hill, London, UK.
Eur J Biochem. 1999 Sep;264(2):569-76. doi: 10.1046/j.1432-1327.1999.00671.x.
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.
半乳糖凝集素-3是一种β-半乳糖苷结合蛋白，尽管该蛋白缺乏转入内质网和高尔基体区室并进入经典分泌途径的信号序列，但仍可从许多细胞中分泌出来。先前的研究表明，将仓鼠半乳糖凝集素-3 N端序列的前120个氨基酸残基连接到细胞质蛋白氯霉素乙酰转移酶（CAT）上，可使融合蛋白在CAT蛋白未分泌的条件下从瞬时转染的Cos细胞中快速分泌。在此我们报告，N端逐步截短会逐渐降低融合蛋白的分泌，最终与起始产物相比降至非常低的水平，但在大部分序列被去除之前，分泌并未完全消除。在半乳糖凝集素-3 N端序列97-120位残基中含有内部缺失的突变型CAT融合蛋白也以与起始产物相似的程度分泌，但进一步缺失89-96位残基则消除了可检测到的分泌。对两种具有分泌能力的CAT融合蛋白中序列YP(90)SAP(93)GAY进行脯氨酸到丙氨酸的诱变，大大降低或消除了它们的分泌，而对这些蛋白半乳糖凝集素-3 N端片段中其他位置存在的脯氨酸配对进行类似诱变则没有效果。结果表明，该序列是半乳糖凝集素-3-CAT融合蛋白分泌的一个关键决定因素，由此推断至少对于转染的Cos细胞中的半乳糖凝集素-3是如此。然而，89-96位残基的短序列本身不足以指导CAT融合蛋白的分泌，似乎仅在半乳糖凝集素-3 N端序列的较大部分的背景下才具有活性。
