Additional N-glycosylation and its impact on the folding of intestinal lactase-phlorizin hydrolase

被引:31
作者
Jacob, R [1 ]
Weiner, JR [1 ]
Stadge, S [1 ]
Naim, HY [1 ]
机构
[1] Hannover Sch Vet Med, Dept Physiol Chem, D-30559 Hannover, Germany
关键词
D O I
10.1074/jbc.275.14.10630
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Lactase-phlorizin hydrolase (LPH) is a membrane bound intestinal hydrolase, with an extracellular domain comprising 4 homologous regions, LPH is synthesized as a large polypeptide precursor, pro-LPH, that undergoes several intra- and extracellular proteolytic steps to generate the final brush-border membrane form LPH beta(final). Pro-LPH is associated through homologous domain IV with the membrane through a transmembrane domain. A truncation of 236 amino acids at the (COOH terminus of domain IV (denoted LAC236) does not significantly influence the transport competence of the generated mutant LPH1646MACT (Panzer, P., Preuss, U,, Joberty, G., and Naim, H. Y. (1998) J. Biol. Chem. 273, 13861-13869), strongly suggesting that LAC236 is an autonomously folded domain that links the ectodomain with the transmembrane region. Here, we examine this hypothesis by engineering several N-linked glycosylation sites into LAC236. Transient expression of the cDNA constructs in COS-l cells confirm glycosylation of the introduced sites. The N-glycosyl pro-LPH mutants are transported to the Golgi apparatus at substantially reduced rates as compared with mild-type pro-LPH, Alterations in LAC236 appear to sterically hinder the generation of stable dimeric trypsin-resistant pro-LPH forms. Individual expression of chimeras containing LAC236, the transmembrane domain and cytoplasmic tail of pro-LPH and GFP as a reporter gene (denoted LAC236-GFP) lends strong support to this view: while LAC236-GFP is capable of forming dimers per se, its N-glycosyl variants are not, The data strongly suggest that the LAC236 is implicated in the dimerization process of pro-LPH, most Likely by nucleating the association of the ectodomains of the enzyme.
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页码:10630 / 10637
页数:8
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