Routing and processing of lactase-phlorizin hydrolase in transfected Caco-2 cells.

Human lactase-phlorizin hydrolase (LPH) is a digestive enzyme that is expressed in the small intestinal brush-border membrane. After terminal glycosylation in the Golgi apparatus, the 230-kDa pro-LPH is cleaved into the 160-kDa brush-border LPHbeta and the 100-kDa profragment (LPHalpha). Since LPHbeta is not transport-competent when it is expressed separately from LPHalpha in COS-1 cells, it was suggested that LPHalpha functions as an intramolecular chaperone.

Chimeric measles viruses with a foreign envelope.

Measles virus (MV) and vesicular stomatitis virus (VSV) are both members of the Mononegavirales but are only distantly related. We generated two genetically stable chimeric viruses. In MGV, the reading frames of the MV envelope glycoproteins H and F were substituted by a single reading frame encoding the VSV G glycoprotein; MG/FV is similar but encodes a G/F hybrid in which the VSV G cytoplasmic tail was replaced by that of MV F.

Effectiveness of cisplatin, paclitaxel, and suramin against human malignant mesothelioma xenografts in athymic nude mice.

Background And Objectives: Malignant mesothelioma has a poor prognosis and is refractory to many agents. The antitumor effectiveness of cisplatin, paclitaxel, and suramin as single agents and in combination was evaluated in vivo against four lines of human pleural malignant mesothelioma xenografts in athymic nude mice, including one epithelial type and three fibrosarcomatous.

Methods: After growth of tumors occurred by day 54 or 55, mice were randomized in groups of four each to receive either cisplatin 4 mg/kg intraperitoneally weekly x5, or paclitaxel (Taxol) 12.

Measles viruses with altered envelope protein cytoplasmic tails gain cell fusion competence.

The cytoplasmic tail of the measles virus (MV) fusion (F) protein is often altered in viruses which spread through the brain of patients suffering from subacute sclerosing panencephalitis (SSPE). We transferred the coding regions of F tails from SSPE viruses in an MV genomic cDNA. Similarly, we constructed and transferred mutated tail-encoding regions of the other viral glycoprotein hemagglutinin (H) gene.

Tyrosine-dependent basolateral sorting signals are distinct from tyrosine-dependent internalization signals.

Converting cysteine 543 to tyrosine in the influenza virus hemagglutinin (HA) introduces both a basolateral sorting signal and an internalization signal into the HA cytoplasmic domain. Another HA mutant, HA+8, contains eight additional amino acids at the end of the cytoplasmic domain that include a powerful internalization signal. HA+8 was also sorted efficiently to the basolateral surface of Madin-Darby canine kidney cells.

A mutation in a highly conserved region in brush-border sucrase-isomaltase and lysosomal alpha-glucosidase results in Golgi retention.

A point mutation in the cDNA of human intestinal sucrase-isomaltase has been recently identified in phenotype II of congenital sucrase-isomaltase deficiency. The mutation results in a substitution of glutamine by proline at position 1098 (Q1098P) in the sucrase subunit. Expression of this mutant sucrase-isomaltase cDNA in COS-1 cells results in an accumulation of sucrase-isomaltase in the ER, intermediate compartment and the cis-Golgi cisternae similar to the accumulation in phenotype II intestinal cells.

The apical sorting of lactase-phlorizin hydrolase implicates sorting sequences found in the mature domain.

Polarized transport of proteins is contingent on the presence of specific protein structures or motifs that function as sorting signals. Our model protein to analyze and to identify such signals is that of lactase-phlorizin hydrolase (LPH), a strictly polarized brush border membrane protein of small intestinal epithelial cells. It is synthesized as a large pro-LPH precursor molecule, which is proteolytically processed to yield the mature brush border enzyme (LPHbeta).

Proteolytic processing of human lactase-phlorizin hydrolase is a two-step event: identification of the cleavage sites.

Human lactase-phlorizin hydrolase (EC 3.2.1.

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.

Endocytosis of chimeric influenza virus hemagglutinin proteins that lack a cytoplasmic recognition feature for coated pits.

The influenza virus A/Japan/305/57 hemagglutinin (HA) can be converted from a protein that is essentially excluded from coated pits into one that is internalized at approximately the rate of uptake of bulk membrane by replacing the HA transmembrane and cytoplasmic sequences with those of either of two other glycoproteins (Roth et al., 1986. J.

Secretion of human intestinal angiotensin-converting enzyme and its association with the differentiation state of intestinal cells.

Human angiotensin I-converting enzyme (ACE) exists in intestinal epithelial cells as a membrane-bound (ACEm) and secretory glycoprotein (ACEsec). The electrophoretic mobilities of ACEsec and ACEm on SDS/polyacrylamide gels are similar; the N-deglycosylated ACEsec and ACEm, in contrast, display slight differences in their apparent molecular masses, indicating that the carbohydrate contents of ACEsec and ACEm are different. Moreover, ACEsec is solely N-glycosylated whereas ACEm is N- and O-glycosylated, assessed by lectin binding studies.

Maturation of human intestinal lactase-phlorizin hydrolase: generation of the brush border form of the enzyme involves at least two proteolytic cleavage steps.

Human lactase-phlorizin hydrolase (LPH), a brush border membrane hydrolase of the small intestine, is synthesized as a precursor molecule that undergoes proteolytic cleavage to yield mature LPH (LPHbeta) by a trypsin-like protease (Naim et al., 1987, 1991). Arg868-Ala869 has been previously proposed to be the putative cleavage site for this processing step.

Congenital sucrase-isomaltase deficiency. Identification of a glutamine to proline substitution that leads to a transport block of sucrase-isomaltase in a pre-Golgi compartment.

Congenital sucrase-isomaltase deficiency is an example of a disease in which mutant phenotypes generate transport-incompetent molecules. Here, we analyze at the molecular level a phenotype of congenital sucrase-isomaltase deficiency in which sucrase-isomaltase (SI) is not transported to the brush border membrane but accumulates as a mannose-rich precursor in the endoplasmic reticulum (ER), ER-Golgi intermediate compartment, and the cis-Golgi, where it is finally degraded. A 6-kb clone containing the full-length cDNA encoding SI was isolated from the patient's intestinal tissue and from normal controls.

Survival after early treatment for carbamyl phosphate synthetase (CPS) I deficiency associated with increase of intramitochondrial CPS I.

The basis for the benefit of early treatment in urea-cycle defects might be an increase in intramitochondrial mutant enzyme in hepatocytes in the postnatal period. In two siblings with carbamyl phosphate synthetase I (CPS I) deficiency, immunoreactive CPS I was greatly reduced in the liver and no residual enzyme activity was detectable. The elder child died at age 4 days, before the diagnosis of CPS I deficiency was established, but in the younger child, age 9 months, treatment was initiated on the 2nd day of life when ammonia concentration was moderately increased, and she has survived.

Folding of human intestinal lactase-phlorizin hydrolase.

The folding of human intestinal prolactase-phlorizin hydrolase (pro-LPH) has been analyzed in a cell-free transcription/translation system. In the presence of the thiol oxidant GSSG, disulfide bond formation in pro-LPH can be promoted concomitant with the binding of the molecule to a conformation-specific monoclonal anti-LPH antibody. Under these conditions, pro-LPH does not bind to the molecular chaperone BiP.

Apical and basolateral coated pits of MDCK cells differ in their rates of maturation into coated vesicles, but not in the ability to distinguish between mutant hemagglutinin proteins with different internalization signals.

In polarized epithelial MDCK cells, all known endogenous endocytic receptors are found on the basolateral domain. The influenza virus hemagglutinin (HA) which is normally sorted to the apical plasma membrane, can be converted to a basolateral protein by specific mutations in its short cytoplasmic domain that also create internalization signals. For some of these mutations, sorting to the basolateral surface is incomplete, allowing internalization of two proteins that differ by a single amino acid of the internalization signal to be compared at both the apical and basolateral surfaces of MDCK cells.

Further characterization of the 5'-flanking region of the rat lactase-phlorizin hydrolase gene.

The lactase-phlorizin hydrolase gene is widely used as a marker of intestinal differentiation. Recent evidence demonstrating that transcription plays a major role in the regulation of this gene suggests that study of the 5'-flanking region will allow an understanding of how the expression of this gene is controlled. However, sequence, restriction, and primer extension analysis of a rat genomic clone has revealed that previously published data are incomplete.

The pro-region of human intestinal lactase-phlorizin hydrolase is enzymatically inactive towards lactose.

The pro-region of intestinal lactase-phlorizin hydrolase (LPH alpha) has been proposed to be important for the correct folding of pro-LPH and mature LPH (LPH beta). In this communication, analysis of the catalytic function of the LPH alpha pro-region is presented. Expression of a cDNA encoding LPH alpha in COS-1 cells reveals a polypeptide that does not hydrolyse lactose.

Intracellular folding of tissue-type plasminogen activator. Effects of disulfide bond formation on N-linked glycosylation and secretion.

The addition of N-linked core oligosaccharides to membrane and secretory glycoproteins occurs co-translationally at asparagine residues in the tripeptide sequon Asn-Xaa-Ser/Thr soon after translocation of the nascent polypeptide into the lumen of the endoplasmic reticulum. However, the presence of the sequon does not automatically ensure core glycosylation, as many proteins contain sequons that remain either unglycosylated or glycosylated to a variable extent. To investigate whether intracellular protein folding can influence sequon utilization, we have expressed tissue-type plasminogen activator (t-PA) in cell culture in the presence of mild concentrations of the reducing agent dithiothreitol to prevent co-translational disulfide bond formation in the endoplasmic reticulum.

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.

Expression of lactase-phlorizin hydrolase in sheep is regulated at the RNA level.

Lactase-phlorizin hydrolase (LPH) is expressed on the intestinal brush border and is responsible for the hydrolysis of lactose, the chief sugar in mammalian milk. The enzyme activity of LPH peaks soon after birth in most mammals and declines to much lower levels before adolescence. The molecular basis of this pattern of expression has not been clearly established.

The polymorphic expression of lactase in adults is regulated at the messenger RNA level.

Background/aims: Lactase phlorizin hydrolase (LPH) activity is high in infants but declines 80%-90% before adulthood in most mammals, including humans. However, 95% of whites show autosomal dominant inheritance of a lifelong high lactose digesting capacity (LDC). This study attempted to clarify the molecular mechanism(s) of this phenomenon (posttranslational vs.

Processing and transport of human small intestinal lactase-phlorizin hydrolase (LPH). Role of N-linked oligosaccharide modification.

The effect of glycosylation on the intracellular transport of human intestinal lactase-phlorizin hydrolase (LPH) was investigated by biosynthetic labeling of biopsy samples in the presence or absence of glycosidase inhibitors. In the presence of deoxynojirimycin (dNM) and deoxymannojirimycin (dMM), endo H sensitive LPH glycoforms of M(r) = 135,000 in both cases were produced (LPHdNM and LPHdMM). The LPH glycoform generated in the presence of swainsonine had an apparent molecular mass of 141,000 (LPHSwa) and was partially sensitive to endo H.