Biosynthesis and maturation of lactase-phlorizin hydrolase in the human small intestinal epithelial cells

Abstract

The biosynthesis and maturation of the human intestinal lactase-phlorizin hydrolase (LPH; EC 3.2.1.23-3.2.1.62) has been studied in cultured intestinal biopsies and mucosal explants. Short time pulse labelling revealed on high mannose intermediate of Mr 215,000 which was converted upon endo-beta-N-acetylglucosaminidase H (endo-H) digestion to a polypeptide of Mr 200,000. The brush border form of LPH was revealed after longer pulse periods and has Mr 160,000. It possesses mainly complex oligosaccharide chains and, owing to its partial endo-H sensitivity, at least one chain of the high mannose type. Leupeptin partially inhibited the appearance of the Mr-160,000 polypeptide. Monensin treatment of biopsies resulted in the modification of the Mr-160,000 species to the Mr-140,000 molecule, which was endo-H sensitive. Pulse-chase analysis indicated a slow post-translational processing of the high mannose precursor (Mr 215,000) to yield the mature brush-border form (Mr 160,000) of LPH. Our results further indicate that LPH is synthesized as a single polypeptide precursor which is intracellularly cleaved to yield the mature brush border of LPH. The data presented suggest that this cleavage occurs during the translocation of the molecule across the Golgi complex.

Functional diversity and interactions between the repeat domains of rat intestinal lactase

“…Furthermore the tandem association of domains I and II is required for the LPHα function, suggestive of a role in folding or in the acquisition of the quaternary structure of the mature enzyme. In rat and rabbit, unlike human, LPHα is subject to internal cleavage during precursor maturation [4,5,29,30]. The functional role (if any) of this cleavage is unknown, but might be related to the different pattern of lactase expression observed along the villus axis in human compared with rat and rabbit ; indeed some enterocytes in hypolactasic humans contain LPH mRNA but no protein, whereas other enterocytes express an inactive protein [55][56][57].…”

“…Here we examine the separate roles of the repeat domains comprised within rat intestinal lactase-phlorizin hydrolase (LPH ; EC 3.2.1.62\108), a major digestive enzyme responsible for the metabolism of dietary lactose in mammalian newborns [1]. LPH is synthesized as a single-chain 215-245 kDa precursor comprising four tandemly repeated domains [2][3][4][5][6][7][8]. These show sequence similarity with the type 1 family of glycosyl hydrolases (T1GH family), a family of proteins that includes single-domain enzymes present in micro-organisms (see [9,10] and references therein] and plants [11][12][13], and in the liver of mammals [14].…”

Processing and transport of human small intestinal lactase‐phlorizin hydrolase (LPH)

“…Preparation of brush border membrane vesicles (BBM) and intracellular membranes (IM) from labeled mucosa, ~munop~ipitations, endo-N-,&acetylglucosaminidase F/glycopeptidase F (endo FIGF) (EC 3.2.2.18 and EC 3.5.1.52, Boe~nger-M~nhe~), endive-a~tyl~u-cosaminidase H (EC 3.2.1.96, B~h~nger-Mannheim) treatment were performed according to Naim et al [4,10,11]. Treatment of immunoprecipitates with O-glycanase (EC 3.2.1.97, Boehringer-Mannheim) was essentially as described in [12] with the following modification.…”

“…After labeling, the specimens were washed three times in RPM1 1640 and homogenized at 4°C with a Teflon-glass homogenizer in 1 ml of 25 mM Tris-HCl @H 8.1), 50 mM NaCl, and a cocktail of protease inhibitors containing 1 mM PMSF, 1 pg pepstatin, 5 pg ieu~ptin, 17.4pg Nadine and 1 pg aprotonin (all from Sigma). Processing of the biopsy homogenates for ~unopr~ipitation with monoclonal anti-human LPH (HBB l/909134/74) [Z] and SDSpolyacrylamide gel electrophoresis were performed as described elsewhere [4].…”

“…Studies in a variety of systems have established that LPH is synthesized as a large singlechain precursor (pro-LPH) that undergoes proteolytic cleavage to the final brush border form (LPHd [l-5]. In human intestinal cells [2,4,5] as well as in the pig small intestine [l] cleavage takes place in the Golgi after complex glycosylation of pro-LPH. The role of intracellular cleavage has been recently investigated by transfection of COS-1 cells with a full length LPH cDNA [6].…”