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J Biol Chem, Vol. 275, Issue 14, 10630-10637, April 7, 2000
From the Department of Physiological Chemistry, School of
Veterinary Medicine Hannover, D-30559 Hannover, Germany
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
Additional N-Glycosylation and Its Impact on the
Folding of Intestinal Lactase-phlorizin Hydrolase*
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-1 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 wild-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.
*
This work was supported by Deutsche Forschungsgemeinschaft
(DFG), Bonn, Germany, Grant Na 331/1-2 (to H. Y. N.) and
Sonderforschungsbereich (SFB) 280.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Physiological
Chemistry, School of Veterinary Medicine, Hannover, Bünteweg 17, D-30559 Hannover, Germany. Tel.: 49-511-9538780; Fax: 49-511-9538585; E-mail: hnaim@biochemie.tiho-hannover.de.
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