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Structure, Function, and Regulation of Intestinal Lactase-Phlorizin Hydrolase and Sucrase- Isomaltase in Health and Disease

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Structure, Function, and Regulation of Intestinal Lactase-Phlorizin Hydrolase and Sucrase- Isomaltase in Health and Disease Gastrointestinal Functions, edited by Edgard E. Delvin and Michael J. Lentze. Nestle Nutrition Workshop Series, Pediatric Program, Vol. 46. Nestec Ltd.. Vevey/Lippincott Williams & Wilkins. Philadelphia © 2001. Structure, Function, and Regulation of Intestinal Lactase-Phlorizin Hydrolase and Sucrase- Isomaltase in Health and Disease Hassan Y. Nairn Department of Physiological Chemistry, School of Veterinary Medicine Hannover, Hannover, Germany Carbohydrates are essential constituents of the mammalian diet. They occur as oligosaccharides, such as starch and cellulose (plant origin), as glycogen (animal origin), and as free disaccharides such as sucrose (plant) and lactose (animal). These carbohydrates are hydrolyzed in the intestinal lumen by specific enzymes to mono- saccharides before transport across the brush border membrane of epithelial cells into the cell interior. The specificity of the enzymes is determined by the chemical structure of the carbohydrates. The monosaccharide components of most of the known carbohydrates are linked to each other in an a orientation. Examples of this type are starch, glycogen, sucrose, and maltose. (3-Glycosidic linkages are minor. Nevertheless, this type of covalent bond is present in one of the major and most essential carbohydrates in mammalian milk, lactose, which constitutes the primary diet source for the newborn. The enzymes implicated in the digestion of carbohydrates in the intestinal lumen are membrane-bound glycoproteins that are expressed at the apical or microvillus membrane of the enterocytes (1-3). In this chapter, the structural and biosynthetic features of the most important disaccharidases, sucrase-isomaltase and lac- tase-phlorizin hydrolase, and the molecular basis of sugar malabsorption (i.e., en- zyme deficiencies) are discussed. STRUCTURE, BIOSYNTHESIS, AND POLARIZED SORTING OF INTESTINAL DISACCHARIDASES Lactase-Phlorizin Hydrolase Lactase-phlorizin hydrolase (LPH) (EC 3.2.1.23/62) is an integral membrane glycoprotein of the intestinal brush border membrane. The same polypeptide chain contains two major hydrolytic activities (4): (a) lactase activity, which is responsible for the hydrolysis of the milk sugar lactose, the main carbohydrate in mammalian milk; and (b) phlorizin hydrolase, which digests (3-glycosylceramides, which are 195 196 LACTASE-PHLORIZIN HYDROLASE AND SUCRASE-1SOMALTASE part of the diet of most vertebrates. Drastic reduction in lactase activity is associated with diarrhea and accompanying symptoms on drinking milk in both newborns and adults; a physiologic role of phlorizin hydrolase activity is still unknown (4). Structural Features The LPH protein is encoded by a completmentary DNA (cDNA) that consists of 6,274 base pairs (bp) and corresponds to 17 exons of the LPH gene, which is located on chromosome 2 (5,6). The amino acid sequence deduced from the LPH cDNA consists of 1,927 amino acids. LPH is a type I integral membrane glycoprotein with an NH2-terminal extracellular domain followed by a transmembrane domain composed of 19 hydrophobic amino acids and a COOH-terminal cytoplasmic domain of 26 amino acids (5). Fig. 1 is a schematic representation of the structure and biosynthesis of LPH. The extracellular domain itself consists of an NH2-terminal cleavable signal pep- tide (19 amino acids) necessary for translocation into the endoplasmic reticulum (11). The LPH molecule is highly glycosylated. The primary sequence of the human enzyme contains 15 potential TV-glycosylation sites (the rabbit and rat enzymes pos- sess 14 and 15, respectively). All cloned LPH species have shown that the molecule consists of four highly conserved structural and functional regions. These domains, denoted I-IV, have co-identity of 38% to 55%. The catalytic activity of lactase is localized to glutamine 1273 in the homologous region III, and of phlorizin hydrolase to glutamine 1749 in the homologous region IV (12). Because of the four homologous regions, LPH may have arisen from two subsequent duplications of one ancestral gene (5). An evolutionary and developmental example that lends support to this notion is given by the sequence similarities of LPH and each of its homologous regions with (3-glycosidases from archaebacteria, eubacteria, and fungi. These simi- larities suggest that LPH is a member of a superfamily of 3-glucosidases and p- galactosidases. The procaryotic (3-glycosidases, on the average, are approximately 50 kd in size, which corresponds to approximately one quarter of the size of full- length LPH or roughly to the size of one homologous region (I-IV). Biosynthesis The mode of biosynthesis and processing of LPH is very similar in the species so far investigated (1,13,14). In what follows, an account of the biosynthesis and processing of the human enzyme is provided (Fig. 1). In small intestinal epithelial cells, LPH is synthesized as a single chain, 215-kd pro-LPH precursor that undergoes core mannose-rich 7V-glycosylation while translocating into the endoplasmic reticu- lum. In the endoplasmic reticulum, pro-LPH monomers form homodimers before processing in the Golgi apparatus (15). The homodimerization event constitutes an absolute requirement for a transport-competent configuration of pro-LPH that ena- bles it to leave the endoplasmic reticulum. The transmembrane domain is directly LACTASE-PHLORIZ1N HYDROLASE AND SUCRASE-ISOMALTASE 197 I— domain I —|— domain II—|— domain III domain IV Arg868-Ala669 1st Cleavage 2nd Cleavage ER/ cis-Golgi Golgi/ TSN brush border Cleavage I. Cleavage II. intracellular extracellular by trypsin FIG. 1. Schematic representation of the structure and biosynthesis of pro-lactase-phlorizin hydrolase (pro-LPH) in human small intestinal cells. Some important structural features of pro- LPH in human small intestinal cells compiled from data which employed biosynthetic studies in human small intestinal explants (1,3,6), cDNA cloning (5), and recombinant expression in COS- 1 (7-9) or Madin-Darby canine kidney (MDCK) cells (10). The /V-terminus starts with a cleavable signal sequence (SS) (MetrGlyig) for cotranslational translocation into the endoplasmic reticu- lum (ER). The ectodomain (Ser2o to Thr1882) can be divided into four homologous domains as indicated. The membrane-anchoring domain (MA) extends from Ala1883 to Leu1902 and the cyto- solic tail (CT) from Ser1903 to Phe1927. Two proteolytic cleavage steps of pro-LPH take place: one intracellularly between Arg734 and Leu735 that generates LPHpinitiai, which is sorted to the brush border membrane. The second cleavage step between Arg868 and Ala869 takes place in the intestinal lumen by pancreatic trypsin and generates the brush border mature enzyme, LPH(3finai (8,9). LPH-a is the profragment that plays a role as an intramolecular chaperone in the folding of pro-LPH. implicated in the dimerization process. Elimination of this region results in a cor- rectly folded pro-LPH monomer which, however, does not dimerize and is blocked in the endoplasmic reticulum and ultimately degraded. Dimerization is not only important for efficient transport from the endoplasmic reticulum, it is crucial for the acquisition of enzymatic activity (15). Here again, correct folding of monomeric pro-LPH is not sufficient for a functional protein. During the pathway through the Golgi apparatus en route to the cell surface, the mannose-rich AMinked sugar chains are processed by mannosidases of the tw-Golgi and several types of sugars are added, resulting in a complex glycosylated protein 198 LACTASE-PHLORIZIN HYDROLASE AND SUCRASE-ISOMALTASE of an approximate apparent molecular weight of 230 kd (3,7). Of particular impor- tance, is 0-glycosylation at the Ser or Thr residues in the LPH molecule. This event, which begins in the cis-Golgi apparatus and ends in the trans-Golgi network, leads to an increase in the enzymatic activity of LPH by a factor of 4 (6). Complex glycosylated mature pro-LPH undergoes two proteolytic cleavage steps (8,9). In the first, the large profragment LPH-a is intracellularly eliminated at Arg734/ Leu735, leaving the membrane-bound LPH-pinitial, which extends from Leu735 to Tyr1927. This form is transported to the apical membrane where it is cleaved by t0 pancreatic trypsin at Arg868/Alag69 LPH-pfinai (Alag69-Tyr1927), which is known as mature brush border LPH of 160 kd (1,13,14). This form comprises the functional domains of the enzyme. The possible role played by the intracellular proteolytic cleavage and the large profragment LPH-a in the acquisition of transport competence and enzymatic activity has been investigated by recombinant expression of pro-LPH in heterologous cell systems. Expression of pro-LPH in the Madin-Darby canine kidney cell line (MDCK) has localized the initial cleavage step (pro-LPH to LPH- Pinitiai) to an intracellular compartment at or immediately after the trans-Golgi net- work (10). In another mammalian cell line—the simian virus-transformed COS cells—cleavage does not take place, thus providing a convenient model to address the important question of the role of cleavage in trafficking and in the function of pro-LPH. Recombinant pro-LPH in COS-1 cells is efficiently transported to the cell surface and shows enzymatic activity similar to that of the intestinal mature brush border LPH (7). Consequently, the intracellular proteolytic processing of pro-LPH in intestinal epithelial cells is not essential for the acquisition of transport competence and biological function. Although the mature brush border form of LPH, the LPH-pfinai species, has been extensively
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