Further studies of glycosylation and intracellular transport of lactase–phlorizin hydrolase in rat small intestine

Abstract

Previous studies [Büller, Montgomery, Sasak & Grand (1987) J. Biol. Chem. 262, 17206-17211] have demonstrated that lactase-phlorizin hydrolase is inserted into the microvillus membrane (MVM) as a large precursor of approx. 220 kDa, which then undergoes two proteolytic cleavage steps to become the 130 kDa mature MVM protein. In order to assess the role of glycosylation in intracellular transport, the processing of this enzyme has been studied in the presence of castanospermine, an inhibitor of N-linked oligosaccharide modification and subsequent treatment with two endoglycosidases, endo-beta-N-acetyl-glucosaminidase (endo-H) and peptide:N-glycosidase-F (N-glycanase). We now show that the intracellular precursor (205 kDa) undergoes carbohydrate processing (220 kDa) and transport to the MVM where its further proteolytic cleavage is as described. Treatment of the intracellular 205 kDa precursor with either endo-H which cleaves only high-mannose N-linked oligosaccharides, or with N-glycanase, which cleaves both high-mannose and complex N-linked oligosaccharides, results in the conversion of the 205 kDa protein band to one of 195 kDa. These data suggest that the 205 kDa precursor contains only high-mannose N-linked carbohydrates, and that the unglycosylated nascent protein is 195 kDa. In the presence of castanospermine, an intracellular precursor of approx. 210 kDa is observed. When treated with endo-H or N-glycanase, this form also produces a protein of 195 kDa. The transport of the intracellular precursor to the MVM and further proteolytic processing is not blocked by the inhibitor. However, all MVM forms of lactase-phlorizin hydrolase show an increase of approx. 5 kDa. Treatment of these three MVM forms with endo-H indicates the increased presence of high mannose oligosaccharides in comparison with non-castanospermine-treated forms. The susceptibility to endo-H of the 130 kDa MVM band synthesized in the absence of castanospermine implies the presence of high-mannose N-linked oligosaccharides in the mature form of lactase-phlorizin hydrolase. Incubation of these MVM forms with N-glycanase further reduces their electrophoretic mobility, indicating the presence of complex N-linked oligosaccharides in the MVM forms, in contrast with the intracellular precursor. Altered glycosylation reduces but does not abolish intracellular transport of lactase-phlorizin hydrolase to the MVM.

Analysis of lactase processing in rabbit

“…In fact this drug caused the appearance of a malglycosylated precursor form that was partially endo-H-sensitive and that was converted into 180 and 150 kDa mature forms. This result is in line with results of previous studies [30] showing that after incubation with castanospermine LPH is still transported to the brush border membrane and proteolytically converted to the mature form. However, the labeled incorporation into LPH proteins was reduced after incubation with castanospermine whereas MDNM treatment did not reduce the lactase biosynthesis in our chase experiments (Fig.…”

Immunoelectron Microscopic Localization of Lactase-Phlorizin Hydrolase in Rat Small Intestine.

“…Plasma mem brane proteins of eukaryotic cell are commonly synthesized and assembled in the rough endoplasmic reticulum, pass through the Golgi apparatus, and are subse quently transferred to the cell surface (2)(3)(4). Previous studies on the biosynthesis and precursor forms of LPH demonstrated that LPH is initially synthesized as a large precursor protein (5)(6)(7)(8). Molecular cloning of pre-LPH cDNA revealed that approximately 45% of amino acid residues in pre-LPH is lost before the mature form of LPH reaches the microvillous membrane (9).…”

“…However, rat LPH might differ from those of human and pig in terms of post-translational processing of precursors. Buller et al (8) suggested that a newly synthesized precursor of LPH in rat jejunum is transported to the BBM, where two subsequent cleavages occur, resulting in the accumulation of mature forms of LPH (13 kDa). In human and pig small intestine, the cleavage of pre-LPH to the mature form of LPH was reported to be leupeptin sensitive, suggesting an intracellular processing, and the only form in the BBM was the mature 160 kDa form (5-7).…”

Suckling Rat Colon Synthesizes and Processes Active Lactase-Phlorizin Hydrolase Immunologically Identical to that from Jejunum

“…Antibodies. The MAb (13) used in these experiments is the same as described previously (12). The hybridoma cells (YBB 2/ 61) were a generous gift of Dr. Andrea Quaroni of Cornell University, Ithaca, NY.…”

“…In our study, we have used techniques established in our laboratory in the study of suckling jejunum (12) to investigate the biosynthesis and function of lactase-phlorizin hydrolase in the early postnatal rat colon. To the best of our knowledge, there has been no report describing the biosynthesis and demonstrating the presence of an active lactase-phlorizin hydrolase enzyme in the colon of newborn animals or human.…”