Hypomorphic variants of lactase-phlorizin hydrolase in congenital lactase deficiency are trafficking incompetent and functionally inactive.

Patients with the rare autosomal recessive disorder congenital lactase deficiency (CLD) present with severe, potentially life-threatening symptoms shortly after birth. Several variants have been characterized within the gene for lactase-phlorizin hydrolase (LCT) that are associated with CLD. Here, we analyze at the biochemical and cellular levels LCT mutants harboring the genetic variants p.Y1390*, p.E1612*, p.S1150Pfs*19, p.S1121L, p.R1587H, and p.S688P. Our data unequivocally demonstrate that these mutants are absolutely transport incompetent, some of which are readily degraded, and are enzymatically inactive. The current study contributes to and expands our understanding on the pathogenesis of CLD at the molecular level.

Structural determinants for transport of lactase phlorizin-hydrolase in the early secretory pathway as a multi-domain membrane glycoprotein.

BACKGROUND: Lactase phlorizin-hydrolase (LPH) is a membrane anchored type I glycoprotein of the intestinal epithelium that is composed of four homologous structural domains. The role of each distinct domain in the intramolecular organization and function of LPH is not completely understood. METHODS: Here, we analyzed the early events of LPH biosynthesis and trafficking by directed restructuring of the domain compositions. RESULTS: Removal of domain I (LPH∆1) results in a malfolded ER-localized protein. By contrast, LPH without domain II (LPH∆2) is normally transported along the secretory pathway, but does not dimerize nor is enzymatically active. Interestingly a polypeptide stretch in domain II between L735-R868 exerts an intriguing role in modulating the trafficking behavior of LPH and its biological function. In fact, association of this stretch with transport-competent LPH chimeras results in their ER-arrest or aberrant trafficking. This stretch harbors a unique N-glycosylation site that is responsible for LPH retention in the ER via association with calnexin and facilitates proper folding of domains I and III before ER exit of LPH. Notably, a similar N-glycosylation site is also found in domain IV with comparable effects on the trafficking of LPH-derived molecules. CONCLUSIONS: Our study provides novel insights into the intramolecular interactions and the sequence of events involved in the folding, dimerization and transport of LPH. GENERAL SIGNIFICANCE: Elucidation of the structural-functional relevance of the domains in pro-LPH is crucial in unravelling and understanding the molecular basis of carbohydrate malabsorption disorders that are associated with lactase deficiency or lactase malfunction.

Congenital lactose intolerance is triggered by severe mutations on both alleles of the lactase gene.

BACKGROUND: Congenital lactase deficiency (CLD) is a rare severe autosomal recessive disorder, with symptoms like watery diarrhea, meteorism and malnutrition, which start a few days after birth by the onset of nursing. The most common rationales identified for this disorder are missense mutations or premature stop codons in the coding region of the lactase-phlorizin hydrolase (LPH) gene. Recently, two heterozygous mutations, c.4419C > G (p.Y1473X) in exon 10 and c.5387delA (p.D1796fs) in exon 16, have been identified within the coding region of LPH in a Japanese infant with CLD. METHODS: Here, we investigate the influence of these mutations on the structure, biosynthesis and function of LPH. Therefore the mutant genes were transiently expressed in COS-1 cells. RESULTS: We show that both mutant proteins are mannose-rich glycosylated proteins that are not capable of exiting the endoplasmic reticulum. These mutant proteins are misfolded and turnover studies show that they are ultimately degraded. The enzymatic activities of these mutant forms are not detectable, despite the presence of lactase and phlorizin active sites in the polypeptide backbone of LPH-D1796fs and LPH-Y1473X respectively. Interestingly, wild type LPH retains its complete enzymatic activity and intracellular transport competence in the presence of the pathogenic mutants suggesting that heterozygote carriers presumably do not show symptoms related to CLD. CONCLUSIONS: Our study strongly suggests that the onset of severe forms of CLD is elicited by mutations in the LPH gene that occur in either a compound heterozygous or homozygous pattern of inheritance.

Structural hierarchy of regulatory elements in the folding and transport of an intestinal multidomain protein.

Human intestinal lactase-phlorizin hydrolase, LPH, encompasses four homologous domains, which presumably have evolved from two subsequent duplications of one ancestral gene. The profragment, LPHalpha, comprises homologous domains I and II and functions as an intramolecular chaperone in the context of the brush-border LPHbeta region of LPH. Here, we analyze the inter-relationship between homologous domains III and IV of LPHbeta and their implication in the overall structure, function, and trafficking of LPH. In silico analyses revealed potential domain boundaries for these domains as a basis for loop-out mutagenesis and construction of deletion or individual domain forms of LPH. Removal of domain IV, which contains lactase, results in a diminished phlorizin hydrolase activity, lack of dimerization in the endoplasmic reticulum (ER), but accelerated transport kinetics from the ER to the Golgi apparatus. By contrast, deletion of domain III, which harbors phlorizin hydrolase, generates a malfolded protein that is blocked in the ER. Interestingly, homologous domain III is transport-competent per se and sorted to the apical membrane in polarized Madin-Darby canine kidney cells. Nevertheless, it neither dimerizes nor acquires complete phlorizin hydrolase activity. Our data present a hierarchical model of LPH in which the homologous domain III constitutes (i) a fully autonomous core domain within LPH and (ii) another intramolecular chaperone besides the profragment LPHalpha. Nevertheless, the regulation of the trafficking kinetics and activity of domain III and entire LPH including elevation of the enzymatic activities require the correct dimerization of LPH in the ER, an event that is accomplished by the non-autonomous domain IV.

Hypolactasia as a molecular basis of lactose intolerance.

Lactase-phlorizin hydrolase (LPH), a membrane-bound glycoprotein present in the luminal surface of enterocytes in the intestine is responsible for lactose intolerance, a phenomenon prevalent in humans worldwide. In the rodent intestine, the post-natal development of the LPH follows a specific pattern, such that the enzyme levels are high in the peri-natal period, but declines considerably upon maturation. The observed maturational decline in the LPH activity is very similar to adult-type hypolactasia observed in humans. Majority of the studies have been carried out using animal models or cell lines and a number of hypotheses have been put forward to explain the maturational decline of lactase activity such as: (a) decreased amount of lactase protein, (b) defect in post-translational modification of precursor lactase to the mature enzyme, and (c) synthesis of an inactive, high molecular weight lactase with altered glycosylation, however, the precise underlying mechanism of adult-type hypolactasia remains undefined. The present review describes the recent developments in understanding the regulation of lactase expression and the possible mechanism of adult-type hypolactasia, as a cause of lactose intolerance.

A novel type of detergent-resistant membranes may contribute to an early protein sorting event in epithelial cells.

One sorting mechanism of apical and basolateral proteins in epithelial cells is based on their solubility profiles with Triton X-100. Nevertheless, apical proteins themselves are also segregated beyond the trans-Golgi network by virtue of their association or nonassociation with cholesterol/sphingolipid-rich microdomains (Jacob, R., and Naim, H. Y. (2001) Curr. Biol. 11, 1444-1450). Therefore, extractability with Triton X-100 does not constitute an absolute criterion of protein sorting. Here, we investigate the solubility patterns of apical and basolateral proteins with other detergents and demonstrate that the mild detergent Tween 20 is adequate to discriminate between apical and basolateral proteins during early stages in their biosynthesis. Although the mannose-rich forms of the apical proteins sucrase-isomaltase, lactase-phlorizin hydrolase, aminopeptidase N, and dipeptidylpeptidase IV reveal similar solubility profiles comprising soluble and nonsoluble fractions, the basolateral proteins, vesicular stomatitis virus G protein, major histocompatibility complex class I, and CD46 are entirely soluble with this detergent. The insoluble Tween 20 membranes are enriched in phosphatidylinositol and phosphatidylglycerol compatible with their synthesis in the endoplasmic reticulum and the existence of a novel class of detergent-resistant membranes. The association of the mannose-rich biosynthetic forms of the apical proteins, sucraseisomaltase, lactase-phlorizin hydrolase, aminopeptidase N, and dipeptidylpeptidase IV with the Tween 20-resistant membranes suggests an early polarized sorting mechanism prior to maturation in the Golgi apparatus.

Partial amino acid sequence and mRNA analysis of cytosolic pyridoxine-beta-D-glucoside hydrolase from porcine intestinal mucosa: proposed derivation from the lactase-phlorizin hydrolase gene.

We have previously identified and purified a novel beta-glucosidase, designated PNGH (pyridoxine-5'-beta-D-glucoside hydrolase), from the cytosolic fraction of pig intestinal mucosal. PNGH catalyses the hydrolysis of PNG (pyridoxine-5'-beta-D-glucoside), a plant derivative of vitamin B6 that exhibits partial nutritional bioavailability in humans and animals. Preliminary amino acid sequence analysis indicated regions of close similarity of PNGH to the precursor form of LPH (lactase-phlorizin hydrolase), the beta-glucosidase localized to the brush-border membrane. We report in the present study amino acid sequence data for PNGH and results of Northern blot analyses, upon which we propose a common genomic origin of PNGH and LPH. Internal Edman sequencing of the PNGH band isolated by SDS/PAGE yielded data for 16 peptides, averaging 10.8 amino acids in length. These peptides from PNGH (approx. 140 kDa) were highly similar to sequences existing over most of the length of the >200 kDa precursor of rabbit LPH; however, we found no PNGH sequences that corresponded to approx. 350 amino acids between positions 463 and 812 of the LPH precursor, a region encoded by exon 7 of the LPH precursor gene (amino acids 568-784), and no sequences that corresponded to regions near the N-terminus. MS analysis of tryptic peptides yielded 25 peptides, averaging 15 amino acids, with masses that matched segments of the rabbit LPH precursor. Northern blot analysis of pig and human small intestinal polyadenylated mRNA using a non-specific LPH cDNA probe showed an expected approx. 6 kb transcript of the LPH precursor, but also an approx. 4 kb transcript that was consistent with the size predicted from the PNGH protein mass. Using a probe specific to the region encoded by exon 7, hybridization occurred only with the 6 kb transcript. Based on these observations, we propose that both PNGH and LPH enzymes have the same genomic origin, but differ in transcriptional and, possibly, post-translational processing.

Annexin II is required for apical transport in polarized epithelial cells.

The sorting of apical proteins comprises an initial recognition step in the trans Golgi network and a final partitioning of the apical pool of proteins into at least two different types of vesicular carriers. One criteria of these carriers is the association or non-association of the protein content with lipid rafts. We have previously characterized a population containing the raft-associated sucrase-isomaltase-carrying vesicles (SAVs) and another one, the non-raft-associated lactase-phlorizin hydrolase-carrrying vesicles (LAVs) that are targeted separately to the apical membrane. Here, we demonstrate biochemically and by employing confocal laser microscopy that the annexin II-S100A10 complex is a component of SAVs and is absent from LAVs. The unequivocal role of annexin II in the apical targeting of SI is clearly demonstrated when down-regulation of this protein by annexin II-specific small interfering RNA drastically decreases the apical delivery of SI in the epithelial cell line Madin-Darby canine kidney. The annexin II-S100A10 complex plays therefore a crucial role in routing SAVs to the apical membrane of epithelial cells.

Enzymatic hydrolysis of pyridoxine-5'-beta-D-glucoside is catalyzed by intestinal lactase-phlorizin hydrolase.

An obligatory step in the mammalian nutritional utilization of pyridoxine-5'-beta-D-glucoside (PNG) is the intestinal hydrolysis of its beta-glucosidic bond that releases pyridoxine (PN). This laboratory previously reported the purification and partial characterization of a novel cytosolic enzyme, designated PNG hydrolase, which hydrolyzed PNG. An investigation of the subcellular distribution of intestinal PNG hydrolysis found substantial hydrolytic activity in the total membrane fraction, of which 40-50% was localized to brush border membrane. To investigate the possible role of a brush border beta-glucosidase in the hydrolysis of PNG, lactase phlorizin hydrolase (LPH) was purified from rat small intestinal mucosa. LPH hydrolyzed PNG with a K(m) of 1.0 +/- 0.1 mm, a V(max) of 0.11 +/- 0.01 micromol/min.mg protein, and a k(cat) of 1.0 s(-1). LPH-catalyzed PNG hydrolysis was inhibited by glucose, lactose, and cellobiose but not by PN. Specific blockage of the phlorizin hydrolase site of LPH using 2',4'-dintrophenyl-2-fluoro-2-deoxy-beta-D-glucopyranoside did not reduce PNG hydrolysis. Evidence of transferase activity was also obtained. Reaction mixtures containing LPH, PNG, and lactose yielded the formation of another PN derivative that was identified as a pyridoxine disaccharide. These results indicate that LPH may play an important role in the bioavailability of PNG, but further characterization is needed to assess its physiological function.

Adsorptive immobilization of intestinal brush border membrane on Triton X-100-substituted Sepharose 4B.

Triton X-100-substituted Sepharose 4B (Sepharose-TX) was used for adsorptive immobilization of intestinal brush border membrane using lactose-phlorizin hydrolase as a representative membrane enzyme. Limited heating of membrane preparations was found to enhance binding. This enhancement is concluded to be owing to a greater availability of the hydrophobic sites, as also confirmed by the 1-anilino-8-naphthalene sulfonate fluorescence studies, for interaction with Triton X-100 moieties on the support. The immobilized preparations obtained by this procedure were found useful in hydrolysis of lactose, involving lactose-phlorizin hydrolase, in continuous operations. It is suggested that the approach may be of general utility for immobilization of biologic membranes by interaction of their extramembrane structures using supports with appropriate hydrophobic groups.

The prosequence of human lactase-phlorizin hydrolase modulates the folding of the mature enzyme.

The efficient transport of proteins along the secretory pathway requires that the polypeptide adopts a stably folded conformation to egress the endoplasmic reticulum (ER). The transport-competent precursor of the brush border enzyme LPH, pro-LPH, undergoes an intracellular cleavage process in the trans-Golgi network between Arg(734) and Leu(735) to yield LPH beta(initial). The role of the prodomain comprising the N-terminally located 734 amino acids of pro-LPH, LPH alpha, in the folding events of LPH beta(initial) has been analyzed by the individual expression of both forms in COS-1 cells. Following synthesis at 37 degrees C LPH beta(initial) acquires a misfolded and enzymatically inactive conformation that is degraded by trypsin. A temperature shift to 20 degrees C generates a stable, trypsin-resistant, and enzymatically active LPH beta(initial) indicating that the individual expression of LPH beta(initial) results in a temperature-sensitive conformation. This form interacts at non-permissive temperatures sequentially with the ER chaperones immunoglobulin-binding protein and calnexin resulting in an ER retention. The LPH alpha prodomain resides in the ER when individually expressed. It reveals compact structural features that are stabilized by disulfide bridges. LPH alpha and LPH beta(initial) readily interact with each other upon coexpression, and this interaction appears to trigger the formation of a trypsin-resistant, correctly folded, enzymatically active, and transport-competent LPH beta(initial) polypeptide. These data clearly demonstrate that the proregion of pro-LPH is an intramolecular chaperone that is critically essential in facilitating the folding of the intermediate form LPH beta(initial) in the context of the pro-LPH polypeptide.

Differential mechanism-based labeling and unequivocal activity assignment of the two active sites of intestinal lactase/phlorizin hydrolase.

Milk lactose is hydrolysed to galactose and glucose in the small intestine of mammals by the lactase/phlorizin hydrolase complex (LPH; EC 3.2.1.108/62). The two enzymatic activities, lactase and phlorizin hydrolase, are located in the same polypeptide chain. According to sequence homology, mature LPH contains two different regions (III and IV), each of them homologous to family 1 glycosidases and each with a putative active site. There has been some discrepancy with regard to the assignment of enzymatic activity to the two active sites. Here we show differential reactivity of the two active sites with mechanism-based glycosidase inhibitors. When LPH is treated with 2',4'-dinitrophenyl 2-deoxy-2-fluoro-beta-D-glucopyranoside (1) and 2', 4'-dinitrophenyl-2-deoxy-2-fluoro-beta-D-galactopyranoside (2), known mechanism-based inhibitors of glycosidases, it is observed that compound 1 preferentially inactivates the phlorizin hydrolase activity whereas compound 2 is selective for the lactase active site. On the other hand, glycals (D-glucal and D-galactal) competitively inhibit lactase activity but not phlorizin hydrolase activity. This allows labeling of the phlorizin site with compound 1 by protection with a glycal. By differential labeling of each active site using 1 and 2 followed by proteolysis and MS analysis of the labeled fragments, we confirm that the phlorizin hydrolysis occurs mainly at the active site located at region III of LPH and that the active site located at region IV is responsible for the lactase activity. This assignment is coincident with that proposed from the results of recent active-site mutagenesis studies [Zecca, L., Mesonero, J.E., Stutz, A., Poiree, J.C., Giudicelli, J., Cursio, R., Gloor, S.M. & Semenza, G. (1998) FEBS Lett. 435, 225-228] and opposite to that based on data from early affinity labeling with conduritol B epoxide [Wacker, W., Keller, P., Falchetto, R., Legler, G. & Semenza, G. (1992) J. Biol. Chem. 267, 18744-18752].

Additional N-glycosylation and its impact on the folding of intestinal lactase-phlorizin hydrolase.

Lactase-phlorizin hydrolase (LPH) is a membrane bound intestinal hydrolase, with an extracellular domain comprising 4 homologous regions. LPH is synthesized as a large polypeptide precursor, pro-LPH, that undergoes several intra- and extracellular proteolytic steps to generate the final brush-border membrane form LPHbeta(final). Pro-LPH is associated through homologous domain IV with the membrane through a transmembrane domain. A truncation of 236 amino acids at the COOH terminus of domain IV (denoted LAC236) does not significantly influence the transport competence of the generated mutant LPH1646MACT (Panzer, P., Preuss, U., Joberty, G., and Naim, H. Y. (1998) J. Biol. Chem. 273, 13861-13869), strongly suggesting that LAC236 is an autonomously folded domain that links the ectodomain with the transmembrane region. Here, we examine this hypothesis by engineering several N-linked glycosylation sites into LAC236. Transient expression of the cDNA constructs in COS-1 cells confirm glycosylation of the introduced sites. The N-glycosyl pro-LPH mutants are transported to the Golgi apparatus at substantially reduced rates as compared with wild-type pro-LPH. Alterations in LAC236 appear to sterically hinder the generation of stable dimeric trypsin-resistant pro-LPH forms. Individual expression of chimeras containing LAC236, the transmembrane domain and cytoplasmic tail of pro-LPH and GFP as a reporter gene (denoted LAC236-GFP) lends strong support to this view: while LAC236-GFP is capable of forming dimers per se, its N-glycosyl variants are not. The data strongly suggest that the LAC236 is implicated in the dimerization process of pro-LPH, most likely by nucleating the association of the ectodomains of the enzyme.

Dietary flavonoid and isoflavone glycosides are hydrolysed by the lactase site of lactase phlorizin hydrolase.

Lactase phlorizin hydrolase (LPH; EC 3.2.1.62) is a membrane-bound, family 1 beta-glycosidase found on the brush border of the mammalian small intestine. LPH, purified from sheep small intestine, was capable of hydrolysing a range of flavonol and isoflavone glycosides. The catalytic efficiency (k(cat)/K(m)) for the hydrolysis of quercetin-4'-glucoside, quercetin-3-glucoside, genistein-7-glucoside and daidzein-7-glucoside was 170, 137, 77 and 14 (mM(-1) s(-1)) respectively. The majority of the activity occurred at the lactase and not phlorizin hydrolase site. The ability of LPH to deglycosylate dietary (iso)flavonoid glycosides suggests a possible role for this enzyme in the metabolism of these biologically active compounds.

Species differences in the sites of cleavage of pro-lactase to lactase supports lack of selective pressure.

The pro-sequences in pro-lactase-phlorizin hydrolase (LPH) are needed for lactase to proceed past the ER, but are irrelevant as to the enzymatic activities. Hence, in all species removal of the pro- sequences (or most of them) must take place after the ER. Contrary to this, the details of the removal of these pro-sequences are to be expected to differ in the various species, since they are not subjected to selective pressure. Using site-directed mutagenesis we investigated processing in rabbit. The first cleavage occurs by furin (or furin-like PCs) and takes place at R-A-A-R(349) in the pro-sequence, generating the known 180 kDa intermediate. Replacing R(349) by Q results in a mutant which is not cleaved but nevertheless transported to the cell surface as demonstrated by immunofluorescence. Further processing of either the 180 kDa intermediate or the mutant is not directly mediated by furin-like PCs, but involves (also) other proteases. These results demonstrate that formation of the 180 kDa intermediate, consistently found only in rabbits, but not in man, is not essential for lactase transport: in all likelihood lack of selective pressure has led to species-specific processing of pro-LPH.

Intestinal lactase-phlorizin hydrolase (LPH): the two catalytic sites; the role of the pancreas in pro-LPH maturation.

Brush border lactase-phlorizin hydrolase carries two catalytic sites. In the human enzyme lactase comprises Glu-1749, phlorizin hydrolase Glu-1273. The proteolytic processing of pro-lactase-phlorizin hydrolase by (rat) enterocytes stops two amino acid residues short of the N-terminus of 'mature' final, brush border lactase-phlorizin hydrolase. Only these two amino acid residues are removed by luminal pancreatic protease(s), probably trypsin.

Protein domains implicated in intracellular transport and sorting of lactase-phlorizin hydrolase.

The roles of various domains of intestinal lactase-phlorizin hydrolase (pro-LPH) on its folding, dimerization, and polarized sorting are investigated in deletion mutants of the ectodomain fused or not fused with the membrane-anchoring and cytoplasmic domains (MACT). Deletion of 236 amino acids immediately upstream of MACT has no effect on the folding, dimerization, transport competence, or polarized sorting of the mutant LPH1646MACT. By contrast, LPH1646, an anchorless counterpart of LPH1646MACT, is not transported beyond the ER and persists as a mannose-rich monomer during its entire life cycle. The further deletion of 87 amino acids generates a correctly folded but transport-incompetent monomeric LPH1559MACT mutant. The results strongly suggest that dimerization and transport of pro-LPH implicate a stretch of 87 amino acids in the ectodomain between LPH1646MACT and LPH1559MACT. In addition, dimerization of pro-LPH requires at least two further criteria: (i) a correctly folded ectodomain of pro-LPH and (ii) the presence of the transmembrane region. Neither of these requirements alone is sufficient for dimerization. Finally, the sorting of pro-LPH appears to be mediated by signals located between the cleavage site of pro-LPH and the LPH1646MACT mutant.

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

Lactase-phlorizin hydrolase (LPH), a major digestive enzyme in the small intestine of newborns, is synthesized as a high-molecular-mass precursor comprising four tandemly repeated domains. Proteolytic cleavage of the precursor liberates the pro segment (LPHalpha) corresponding to domains I and II and devoid of known enzymic function. The mature enzyme (LPHbeta) comprises domains III and IV and is anchored in the brush border membrane via a C-terminal hydrophobic segment. To analyse the roles of the different domains of LPHalpha and LPHbeta, and the interactions between them, we have engineered a series of modified derivatives of the rat LPH precursor. These were expressed in cultured cells under the control of a cytomegalovirus promoter. The results show that recombinant LPHbeta harbouring both domains III and IV produces lactase activity. Neither domain III nor IV is alone sufficient to generate active enzyme, although the corresponding proteins are transport-competent. Tandem duplication of domains III or IV did not restore lactase activity, demonstrating the separate roles of both domains within LPHbeta. Further, the development of lactase activity did not require LPHalpha; however, LPHalpha potentiated the production of active LPHbeta but the individual LPHalpha subdomains I and II were unable to do so. Lactase activity and targeting required the C-terminal transmembrane anchor of LPH; this requirement was terminal transmembrane anchor or LPH; this requirement was not satisfied by the signal/anchor region of another digestive enzyme: sucrase-isomaltase. On the basis of this study we suggest that multiple levels of intramolecular interactions occur within the LPH precursor to produce the mature enzyme, and that the repeat domains of the precursor have distinct and specific functions in protein processing, substrate recognition and catalysis. We propose a functional model of LPHbeta in which substrate is channelled from an entry point located within domain II to the active site located in domain IV.

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). Pro-LPH as well as LPHbeta are correctly sorted to the brush border membrane. In this paper we examine the location of putative sorting signals in the pro-LPH molecule. Expression of a cDNA encoding the LPHbeta mature form in the absence of the LPHalpha species in Madin-Darby canine kidney (MDCK) cells reveal an LPHbeta molecule that is not as transport-competent as wild type pro-LPH. The proportion of complex glycosylated LPHbeta constitutes not more than 10% of the total synthesized protein. This form displays a similar trypsin sensitive pattern as wild type intestinal LPHbeta suggesting comparable folding patterns of the two species. Complex glycosylated LPHbeta is sorted to the apical membrane more efficiently than wild type pro-LPH. We conclude that the apical sorting signals for pro-LPH are exclusively found in the LPHbeta mature domain.

Lactase phlorhizin hydrolase turnover in vivo in water-fed and colostrum-fed newborn pigs.

We have estimated the synthesis rates in vivo of precursor and brush-border (BB) polypeptides of lactase phlorhizin hydrolase (LPH) in newborn pigs fed with water or colostrum for 24h post partum. At the end of the feeding period, piglets were anaesthetized and infused intravenously for 3h with L-[4-3H]- phenylalanine. Blood and jejunal samples were collected at timed intervals. The precursor and BB forms of LPH were isolated from jejunal mucosa by immunoprecipitation followed by SDS/PAGE, and their specific radioactivity in Phe determined. The kinetics of precursor and BB LPH labelling were analysed by using a linear compartmental model. Immunoisolated LPH protein consisted of five polypeptides [high-mannose LPH precursor (proLPHh), complex glycosylated LPH precursor (proLPHe), intermediate complex glycosylated LPH precursor (proLPH1i) and two forms of BB LPH]. The fractional synthesis rate (Ks) of proLPHh and proLPHc (approx. 5%/min) were the same in the two groups but the absolute synthesis rate (in arbitrary units, min-1) of proLPHh in the colostrum-fed animals was twice that of the water-fed animals. The Ks values of proLPHi polypeptides were significantly different (water-fed, 3.89%/min; colostrum-fed, 1.6%/min), but the absolute synthesis rates did not differ. The Ks of BB LPH was not different between experimental treatment groups (on average 0.037%/min). However, the proportion of newly synthesized proLPHh processed to BB LPH was 48% lower in colostrum-fed than in water-fed animals. We conclude that in neonatal pigs, the ingestion of colostrum stimulates the synthesis of proLPHh but, at least temporarily, disrupts the processing of proLPH polypeptides to the BB enzyme.