Dimerization of lactase-phlorizin hydrolase occurs in the endoplasmic reticulum, involves the putative membrane spanning domain and is required for an efficient transport of the enzyme to the cell surface.

Analysis of the quaternary structure of human intestinal lactase-phlorizin hydrolase (LPH) by chemical cross-linking and sucrose-gradient centrifugation reveals that the brush border form of LPH (LPH beta; 160-kDa) is a homodimeric molecule. Dimerization ensures in the ER when LPH is still exclusively found as an uncleaved mannoserich precursor (pro-LPHb; 215-kDa). This is supported by the following observations. (i) Biosynthetically labeled intestinal biopsy specimens as well as transfected COS-1 cells expressing pro-LPH contain monomeric and dimeric forms of pro-LPHb; the complex glycosylated pro-LPH (pro-LPHc; 230-kDa) as well as the cleaved mature LPH beta species in intestinal biopsy samples are discerned exclusively as dimers. (ii) Dimeric forms of pro-LPHh could be also detected when cells were biosynthetically labeled at 15 degrees C, at which temperature the egress of pro-LPH from the ER is blocked. Dimerization is essential for the transport competence of pro-LPH and is strongly associated with the presence of an intact transmembrane domain. Mutant pro-LPH-mact lacking the complete transmembrane domain persists as a monomeric, mannose-rich and transport-incompetent molecule that is not secreted into the exterior milieu, accumulates most likely in the ER and is ultimately degraded. Further, deletion of the cytoplasmic tail in the pro-LPH-ct mutant leads to marked reduction in the proportion of dimeric as well as complex glycosylated pro-LPH-ct. Finally, dimerization is linked to the acquisition of LPH to its biological function, since only dimers of wild type pro-LPH or pro-LPH-ct are enzymatically active, while their monomeric counterparts as well as pro-LPH-mact are not.

The pro region of human intestinal lactase-phlorizin hydrolase.

Human small intestinal lactase-phlorizin hydrolase (LPH) is synthesized as a single-chain polypeptide precursor, prepro-LPH, that undergoes two sequential cleavage steps: the first in the endoplasmic reticulum to pro-LPH (215-kDa) and the second, following terminal glycosylation in the Golgi apparatus, to mature 160-kDa LPH (denoted LPH beta). The LPH beta molecule is subsequently targetted to the brush-border membrane. Characterization of the N-terminal profragment (denoted LPH alpha) of pro-LPH using an epitope-specific, anti-peptide polyclonal antibody reveals that LPH alpha (i) has an apparent molecular weight of approximately 100,000, (ii) is not associated with LPH beta after cleavage of pro-LPH has occurred, and (iii) is not transported to the cell surface or secreted into the extracellular medium. In biosynthetic labeling experiments, a clear precursor/product relationship could be demonstrated between pro-LPH and the LPH alpha and LPH beta polypeptides. Further, LPH alpha has a significantly shorter half-life than LPH beta. LPH alpha is neither N- nor O-glycosylated, despite the presence of 5 potential N-glycosylation sites. LPH alpha, which is rich in cysteine and hydrophobic amino acid residues, may fold rapidly into a tight and rigid globular domain in which carbohydrate attachment sites are no longer accessible to glycosyltransferases. When expressed independently in COS-1 cells, the LPH beta polypeptide forms a misfolded, transport-incompetent molecule. We propose a role for the LPH alpha domain within the pro-LPH molecule as an intramolecular chaperone during folding in the ER.

Biogenesis of intestinal lactase-phlorizin hydrolase in adults with lactose intolerance. Evidence for reduced biosynthesis and slowed-down maturation in enterocytes.

Enzymatic activity, biosynthesis, and maturation of lactasephlorizin hydrolase (LPH) were investigated in adult volunteers with suspected lactose intolerance. Mean LPH activity in jejunal biopsy homogenates of these individuals was 31% compared to LPH-persistent individuals, and was accompanied by a reduced level of LPH-protein. Mean sucrase activity in individuals with low LPH was increased to 162% and was accompanied by an increase in sucrase-isomaltase (SI)-protein. Biosynthesis of LPH, SI, and aminopeptidase N (APN) was studied in organ culture of small intestinal biopsy specimens. In individuals with LPH restriction, the rate of synthesis of LPH was drastically decreased, reaching just 6% of the LPH-persistent group after 20 h of culture, while the rate of synthesis of SI appeared to be increased. In addition, maturation of pro-LPH to mature LPH occurred at a slower rate in LPH-restricted tissue. Immunoelectron microscopy revealed an accumulation of immunoreactive LPH in the Golgi region of enterocytes from LPH-restricted individuals and reduced labeling of microvillus membranes. Therefore, lactose intolerance in adults is mainly due to a decreased biosynthesis of LPH, either at the transcriptional or translational level. In addition, intracellular transport and maturation is retarded in some of the LPH-restricted individuals, and this leads to an accumulation of newly synthesized LPH in the Golgi and a failure of LPH to reach the microvillus membrane.

Human intestinal lactase-phlorizin hydrolase: isolation and preparation of a specific antiserum.

Human intestinal lactase-phlorizin hydrolase (lactase) was selectively isolated with monospecific polyclonal antibodies to rat lactase. In addition to their immunologic similarities indicated by this isolation, human and rat lactase have similar kinetic characteristics but different subunit structure when analyzed by gel electrophoresis under reducing conditions. Rabbits immunized by injecting human lactase complexed with anti-rat lactase produced specific antibodies to human lactase that exhibited little cross-reactivity to the rat enzyme. The simple single-step procedure allows isolation of human lactase in high purity from small biologic samples and preparation of specific antisera to the human enzyme.

Suckling rat colon synthesizes and processes active lactase-phlorizin hydrolase immunologically identical to that from jejunum.

To identify potential tissue-specific characteristics of intestinal glycoprotein synthesis and processing, rat intestinal lactase-phlorizin hydrolase (L-Ph) was studied after pulse-labeling of colonic explants from 5-d-old suckling rats in organ culture and the data compared to similar studies in rat jejunum. Histologic sections of 5-d-old proximal colon showed villus-like structures lined with columnar epithelial cells. Lactase and phlorizin hydrolase activities showed tissue-specific developmental patterns. Using a MAb to small intestinal L-Ph, we were able to immunoprecipitate from colon at different ages a protein that hydrolyzed lactose and phlorizin, and whose activity was not inhibited by p-chloromercuribenzoate. After pulse-labeling for 60 min and chase for 30 min, immunoprecipitated L-Ph from total homogenates of rat colonic explants appeared on fluorography of SDS-PAGE as one band of approximately 205 kD. With increasing time of chase, it took 240 min before the precursor form was converted to the intermediate form (equivalent to the 180-kD form in jejunum) and the mature form (equivalent to the 130-kD form in jejunum), although these conversions in the jejunum were observed within 60 min of chase, and only 30 min of pulse labeling. When compared on SDS-PAGE to immunoprecipitated jejunal L-Ph, the precursor form in the colon had a slightly higher apparent mol wt than the corresponding precursor form found in the endoplasmic reticulum-Golgi fraction of the jejunum. The intermediate as well as the mature L-Ph forms in the colon were also both somewhat higher in apparent molecular weight than the same bands in the microvillus membrane fraction from jejunal explants. Removal of N-linked oligosaccharides from jejunum and colonic forms of L-Ph produced bands on SDS-PAGE with identical mobility, suggesting that the proteins were the same. The data demonstrate that, in neonatal colon, enzymatically active L-Ph undergoes biosynthetic and processing events similar to those in the jejunum. During early life, colonic L-Ph may function in the salvage of lactose not absorbed in the small intestine.