Biomedical subjects

H Y Naim

Publications and source records attributed to H Y Naim.

At least 37 records · Page 2Linked to original sources

PubMed1998

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.

Animals↗

PubMed1998

Analysis of a naturally occurring mutation in sucrase-isomaltase: glutamine 1098 is not essential for transport to the surface of COS-1 cells.

A glutamine for proline substitution at position 1098 was previously shown to result in accumulation of brush-border sucrase-isomaltase in the Golgi apparatus. The substitution is present in a highly homologous region of the protein, and results in a comparable accumulation when introduced into the same region in lysosomal alpha-glucosidase. To study the importance of the glutamine-1098, we analyzed the transport compatibility of two mutants in which glutamine-1098 is substituted by lysine or alanine. Both mutants were transported to the cell surface and processed comparable to wild type. We concluded that glutamine-1098 is not essential for transport to the cell surface.

Amino Acid Sequence↗

PubMed1998

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. What happens to LPHalpha after cleavage is still unclear. To analyze and localize LPHalpha in polarized epithelial cells, wild type and tagged LPH were stably expressed in Caco-2 cells. In tagged LPH, a vesicular stomatitis virus epitope tag was inserted into the LPHalpha region. Wild type and tagged proteins were processed at similar rates, and both cleaved LPHbeta forms were expressed at the apical cell surface. Pro-LPH was recognized by antibodies against LPH, a profragment epitope and the vesicular stomatitis virus tag. LPHalpha alone, however, could not be recovered by these antibodies. Our data suggest that LPHalpha is degraded immediately after cleavage.

Amino Acid Sequence↗

PubMed1998

T-cell independent IgM and enduring protective IgG antibodies induced by chimeric measles viruses.

B-cell activation depends on the intensity of B-cell receptor cross-linking. Studies of haptenated antigens and vesicular stomatitis virus (VSV) have demonstrated a correlation between antigen repetitiveness and the degree to which B-cell activation is independent of T cells. Here, we compare neutralizing antibody responses to inactivated VSV with those to two inactivated human pathogenic viruses: highly cytopathic poliovirus (PV) and poorly cytopathic measles virus (MV). The rigidly structured PV efficiently induced neutralizing IgM antibodies independent of T cells. In contrast, neutralizing antibodies to the pleomorphic MV were dependent on helper T cells. To test whether this resulted from the differences in virus structure or the capacity of MV to induce cell fusion and/or immunosuppression, we analyzed antibody responses to chimeric MV expressing VSV glycoprotein instead of MV fusion protein and hemagglutinin. IgM antibodies were independent of T cells; in addition, we found IgG responses dependent on T-cell help that were enduring and protective against lethal VSV infection. Because chimeric MV viruses look like MV ultrastructurally, we conclude that not only structural differences in the envelope but also the ability of MV to induce immunosuppression may limit its capacity to directly activate B cells. These findings are relevant for our understanding of B-cell activation by two prototypic human pathogenic viruses and for the design of new recombinant vaccines.

Animals↗

PubMed1998

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. From the mutated genomic cDNAs, we achieved rescue of viruses that harbor different alterations of the F tail, deletions in the membrane-distal half of the H tail, and combinations of these mutations. Viruses with alterations in any of the tails spread rapidly through the monolayer via enhanced cell-cell fusion. Double-tail mutants had even higher fusion competence but slightly decreased infectivity. Analysis of the protein composition of released mutant viral particles indicated that the tails are necessary for accurate virus envelope assembly and suggested a direct F tail-matrix (M) protein interaction. Since even tail-altered glycoproteins colocalized with M protein in intracellular patches, additional interactions may exist. We conclude that in MV infections, including SSPE, the glycoprotein tails are involved not only in virus envelope assembly but also in the control of virus-induced cell fusion.

Amino Acid Sequence↗

PubMed1998

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. In contrast to MG/FV, MGV virions do not contain the MV matrix (M) protein. This demonstrates that virus assembly is possible in the absence of M; conversely, the cytoplasmic domain of F allows incorporation of M and enhances assembly. The formation of chimeric viruses was substantially delayed and the titers obtained were reduced about 50-fold in comparison to standard MV. In the novel chimeras, transcription and replication are mediated by the MV ribonucleoproteins but the envelope glycoproteins dictate the host range. Mice immunized with the chimeric viruses were protected against lethal doses of wild-type VSV. These findings suggest that it is feasible to construct MV variants bearing a variety of different envelopes for use as vaccines or for gene therapeutic purposes.

Amino Acid Sequence↗

PubMed1997

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. The simplest explanation for the observation that multiple sorting phenotypes depend upon the same small amino acid sequence is that certain tyrosine-based internalization signals might also function as basolateral sorting signals. To test this hypothesis, second-site mutations were introduced into HA C543Y or HA+8 to determine if the internalization and basolateral sorting functions can be separated. For HA C543Y, the same sequence positions were important for both basolateral sorting and internalization, but the two functions responded differently to individual amino acid replacements, indicating that they were distinct. For HA+8, the basolateral sorting signal required the same tyrosine as the internalization signal, but did not share any other characteristics. Thus, even when basolateral sorting signals that depend on tyrosine overlap or are co-linear with internalizations signals, the two sorting processes are sensitive to different characteristics of the sequence.

Amino Acid Sequence↗

PubMed1997

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. An interesting feature of the Q1098P substitution is its location in a region of the sucrase subunit that shares striking similarities with the isomaltase subunit and other functionally related enzymes, such as human lysosomal acid alpha-glucosidase and Schwanniomyces occidentalis glucoamylase. We speculated that the Q-->P substitution in these highly conserved regions may result in a comparable accumulation. Here we examined this hypothesis using lysosomal alpha-glucosidase as a reporter gene. Mutagenesis of the glutamine residue at position 244 in the homologous region of alpha-glucosidase to proline results in a protein that is neither transported to the lysosomes nor secreted extracellularly but accumulates in the ER, intermediate compartment and cis-Golgi as a mannose-rich polypeptide similar to mutant sucrase-isomaltase in phenotype II. We propose that the Q1098P and Q244P mutations (in sucrase-isomaltase and alpha-glucosidase, respectively) generate structural alterations that are recognized by a control mechanism, operating beyond the ER in the intermediate compartment or cis-Golgi.

Amino Acid Sequence↗

PubMed1997

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.

Animals↗

PubMed1996

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

Human lactase-phlorizin hydrolase (EC 3.2.1.23/62) is a major disaccharidase in the microvillus membrane of small intestinal epithelial cells. The enzyme is synthesized as a single-chain precursor protein and undergoes proteolytic processing during maturation. We studied proteolytic processing of human lactase-phlorizin hydrolase in transfected COS-1, Caco-2, and MDCK cells using metabolic labeling, surface immunoprecipitation, protease sensitivity assays, and microsequencing. Furthermore, we generated mutated forms of the enzyme to alter potential proteolytic cleavage sites and expressed these in Caco-2 and COS-1 cells. Since the N-terminal amino acid of microvillus lactase-phlorizin hydrolase corresponds to Ala869 in the precursor protein, it has been speculated that processing occurs at position Arg868-Ala869. Substitution of Arg868 with isoleucine, lysine, or glutamic acid had no effect on the proteolytic processing of pro-LPH in Caco-2 cells. As in wild-type enzyme a processed 160-kDa form was generated. These data are not consistent with a primary proteolytic processing at position Arg868-Ala869. Using amino-terminal amino acid sequencing of this processed form isolated from stable transfected MDCK cells we identified the cleavage site at Arg734-Leu735. Treatment of pro-lactase-phlorizin hydrolase expressed in COS-1 and MDCK cells by trypsin yielded a 145-kDa form with an identical amino terminal as the mature microvillus enzyme isolated from intestinal mucosa (Ala869). These data provide unambiguous evidence of a two-step processing of human lactase-phlorizin hydrolase. The first cleavage occurs intracellularly after a dibasic site (Arg734-Leu735) and yields the 160-kDa intermediate form. In a second step the intermediate form inserted into the microvillus membrane is trimmed to the mature enzyme by luminal trypsin.

Animals↗

PubMed1996

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. Spontaneous release of ACEsec is achieved by incubation of brush border membranes at 37 degrees C. Aprotinin, leupeptin and EDTA partly inhibit the generation of ACEsec, strongly suggesting that ACEsec is generated from ACEm by proteolytic cleavage. The expression levels of ACEsec in the intestine may be associated with the differentiation state of mucosal cells. Thus ACEsec is more abundant than ACEm in immature non-epithelial crypt cells of patients with coeliac disease. Well-differentiated epithelial cells, by contrast, contain predominantly ACEm. The variations in the proportions of cleaved ACEsec in differentiated and non-differentiated cells may be due to varying levels of the cleaving protease. Alternatively, because epithelial cell differentiation is accompanied by alterations in the levels of oligosaccharyltransferases, the results suggest a critical role for the glycosylation pattern of ACEm in its susceptibility to the putative cleaving protease.

Aprotinin↗

PubMed1996

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. Site-directed mutagenesis of this monobasic site does not lead to the generation of an uncleaved proLPH species, which strongly suggests the existence of an additional cleavage site. Further analyses of LPH synthesized in different cell lines lend support to this hypothesis. Biosynthetic labeling of human intestinal biopsy samples in the presence of trypsin reveals an LPHbeta species that is slightly smaller than the intracellularly cleaved molecule. When the proLPH molecule is screened for potential cleavage sites, two dibasic pairs are revealed upstream of the N-terminal end of brush border LPH at Lys851-Arg852 and Arg830-Lys831. Treatment of proLPH with trypsin for different periods of time supports the idea of at least two cleavage steps, whereby Arg868-Ala869 represents the final cleavage site that generates LPHbeta. We propose that the initial cleavage of proLPH takes place intracellularly at a site further away from Arg868-Ala869, to generate LPHbeta initial; LPHbeta is subsequently cleaved extracellularly in the gut lumen, presumably by trypsin, at Arg868-Ala869 to mature brush border LPH (LPHbeta initial).

Amino Acid Sequence↗

PubMed1996

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. Sequencing of the cDNA revealed a single mutation, A/C at nucleotide 3298 in the coding region of the sucrase subunit of the enzyme complex. The mutation leads to a substitution of the glutamine residue by a proline at amino acid 1098 (Q1098P). The Q1098P mutation lies in a region that is highly conserved between sucrase and isomaltase from different species and several other structurally and functionally related proteins. This is the first report that characterizes a point mutation in the SI gene that is responsible for the transport incompetence of SI and for its retention between the ER and the Golgi.

Amino Acid Sequence↗

PubMed1996

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. Cell Biol. 102:1271-1283). To identify more precisely the foreign amino acid sequences responsible for this change in HA traffic, DNA sequences encoding the transmembrane (TM) or cytoplasmic (CD) domains of either the G glycoprotein of vesicular stomatitis virus (VSV) or the gC glycoprotein of herpes simplex virus were exchanged for those encoding the analogous regions of wild type HA (HA wt). HA-HA-G and HA-HA-gC, chimeras that contain only a foreign CD, resembled HA wt in having a long residence on the cell surface and were internalized very slowly. HA-HA-gC was indistinguishable from HA in our assays, whereas twice as much HA-HA-G was internalized as was HA wt. However, HA-G-HA, containing only a foreign TM, was internalized as efficiently as was HA-G-G, a chimeric protein with transmembrane and cytoplasmic sequences of VSV G protein. Conditions that blocked internalization through coated pits also inhibited endocytosis of the chimeric proteins. Although the external domains of the chimeras were less well folded than that of the wild type HA, denaturation of the wild type HA external domain by treatment with low pH did not increase the interaction of HA with coated pits. However, mutation of four amino acids in the TM of HA allowed the protein to be internalized, indicating that the property that allows HA to escape endocytosis resides in its TM. These results indicate that possession of a cytoplasmic recognition feature is not required for the internalization of all cell surface proteins and suggest that multiple mechanisms for internalization exist that operate at distinctly different rates.

Amino Acid Sequence↗

PubMed1996

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.

Animals↗

PubMed1995

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. Intramitochondrial CPS I was substantially higher in this sibling than in the elder sister. The different outcome in the younger patient was probably attributable to prompt treatment after early diagnosis.

Amino Acid Metabolism, Inborn Errors↗

PubMed1995

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. In the absence of GSSG, on the other hand, pro-LPH does not bind to the monoclonal anti-LPH antibody, but can be immunoprecipitated with a polyclonal antibody that is directed against a denatured form of the enzyme. In this case, interaction of pro-LPH with immunoglobulin heavy chain binding protein can be discerned. The results demonstrate the existence of intramolecular disulfide bonds that are essential for the promotion of pro-LPH to a native conformation. Furthermore, BiP is involved in the folding events of pro-LPH.

Adenosine Triphosphate↗

PubMed1995

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. In the present study, we used a directed sequencing strategy to carefully analyze this region. Our expanded analysis of the 5'-flanking region of the lactase-phlorizin hydrolase gene should facilitate future studies of its structure and function.

Animals↗