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M A Alonso

Publications and source records attributed to M A Alonso.

At least 37 records · Page 2Linked to original sources
PubMed1999

Incorporation of MAL, an integral protein element of the machinery for the glycolipid and cholesterol-mediated apical pathway of transport, into artificial membranes requires neither of these lipid species.

The MAL proteolipid, an integral membrane protein with selective residence in glycolipid- and cholesterol-enriched membrane (GEM) microdomains, has recently been identified as being an element of the integral protein machinery necessary for apical transport in MDCK cells. With the use of a recombinant baculovirus, we have expressed and purified polyhistidine-tagged MAL to determine whether MAL has special lipid requirements for becoming incorporated into membranes. In contrast with caveolin-1, a component of GEMs that requires cholesterol for its integration into artificial membranes, MAL incorporation took place with dimyristoylphosphatidylcholine as the only lipid component. The presence of cholesterol, sphingomyelin, or galactocerebrosides did not affect the efficiency of this process. These results indicated that MAL is compatible with membranes containing either only phospholipids or also glycolipids and cholesterol and are consistent with the reported requirement of a sorting event for the specific targeting of MAL to GEM microdomains.

Animals↗
PubMed1999

The MAL gene is expressed in primary mediastinal large B-cell lymphoma.

Primary mediastinal large B-cell lymphoma (PMBL) appears to be a distinct clinicopathologic entity among diffuse large B-cell lymphomas (DLBLs). To find molecular alterations associated with this disease, we compared the mRNAs expressed in 3 PMBLs and 3 peripheral DLBLs by differential display-reverse transcription (DDRT) and identified a mRNA specifically expressed in PMBLs. Sequence analysis showed that this mRNA is encoded by the MAL gene, the expression of which was shown to be restricted to the T-cell lineage during hematopoiesis. MAL gene expression was demonstrated by Northern blot and reverse transcription-polymerase chain reaction (RT-PCR) in 8 of 12 PMBLs. However, there was little or no MAL gene expression in 8 peripheral DLBLs. Immunohistochemical analysis evidenced expression of MAL protein in tumoral B cells restricted to the PMBL subtype. Finally, Southern blot studies did not demonstrate rearrangement of the MAL gene. Altogether, our results indicate that MAL expression is recurrent in PMBLs, providing further evidence that PMBL represents a distinct entity among DLBLs. Because MAL protein is located in detergent-insoluble glycolipid-enriched membrane (GEM) domains involved in lymphocyte signal transduction, abnormal expression of MAL protein in the B-lymphoid lineage may have significant implications in PMBL lymphomagenesis.

Adult↗
PubMed1999

Substitution of the two carboxyl-terminal serines by alanine causes retention of MAL, a component of the apical sorting machinery, in the endoplasmic reticulum.

MAL, a selective resident of glycolipid-enriched membranes (GEMs), is an integral membrane protein necessary for apical transport and accurate sorting of the influenza virus hemagglutinin in MDCK cells. The carboxyl-terminal end of MAL has the sequence Phe-Ser-Leu-Ile-Arg-Trp-Lys-Ser-Ser (FSLIRWKSS), which includes the LIRW motif necessary for sorting MAL to GEMs, and whose last five amino acids resemble dilysine-based signals involved in endoplasmic reticulum (ER) retention. We have addressed the influence of the carboxyl-terminal serines in both MAL distribution and incorporation into GEMs. Substitution of the serines by alanine impeded the access of MAL to GEMs and changed its distribution from a perinuclear distribution to an ER pattern. The RWKSS sequence appended to the carboxyl-terminus of CD4 caused retention of the chimera in the ER. Thus, although this pentapeptide can function producing ER retention in other protein context, the presence of the carboxyl-terminal serines in the intact MAL molecule prevents its use as an ER-retention signal.

Alanine↗
PubMed1999

The MAL proteolipid is necessary for normal apical transport and accurate sorting of the influenza virus hemagglutinin in Madin-Darby canine kidney cells.

The MAL (MAL/VIP17) proteolipid is a nonglycosylated integral membrane protein expressed in a restricted pattern of cell types, including T lymphocytes, myelin-forming cells, and polarized epithelial cells. Transport of the influenza virus hemagglutinin (HA) to the apical surface of epithelial Madin-Darby canine kidney (MDCK) cells appears to be mediated by a pathway involving glycolipid- and cholesterol- enriched membranes (GEMs). In MDCK cells, MAL has been proposed previously as being an element of the protein machinery for the GEM-dependent apical transport pathway. Using an antisense oligonucleotide-based strategy and a newly generated monoclonal antibody to canine MAL, herein we have approached the effect of MAL depletion on HA transport in MDCK cells. We have found that MAL depletion diminishes the presence of HA in GEMs, reduces the rate of HA transport to the cell surface, inhibits the delivery of HA to the apical surface, and produces partial missorting of HA to the basolateral membrane. These effects were corrected by ectopic expression of MAL in MDCK cells whose endogenous MAL protein was depleted. Our results indicate that MAL is necessary for both normal apical transport and accurate sorting of HA.

Animals↗
PubMed1999

Targeting of MAL, a putative element of the apical sorting machinery, to glycolipid-enriched membranes requires a pre-golgi sorting event.

The MAL proteolipid is a nonglycosylated polytopic membrane protein with specific residence in glycolipid-enriched membrane (GEM) microdomains. MAL has been proposed as an element of the machinery for apical transport in polarized epithelial cells. Previous work demonstrated that MAL requires its four carboxyl-terminal amino acids to be targeted to GEMs. In the present work, we have engineered MAL with N-glycosylation consensus sequences to delimit the site at which commitment of MAL to access into GEMs takes place. Comparison of engineered MAL proteins bearing either an intact or a truncated carboxyl terminus revealed that whereas the former acquired endo H-sensitive and endo H-resistant mature glycosylation, the protein with a deleted carboxyl terminus did not. These results indicate that although MAL incorporation into GEMs takes place mainly in the Golgi, commitment of MAL to enter GEMs is a pre-Golgi event.

Amino Acid Sequence↗
PubMed1999

CD4 segregates into specific detergent-resistant T-cell membrane microdomains.

In T cells, glycolipids, glycoproteins attached to the membrane via a glycosylphosphatidylinositol (GPI) anchor, and Src-like tyrosine kinases are highly enriched in a membrane fraction resistant to solubilization by nonionic detergents. We have investigated the distribution of CD4 in T-cell membranes and found that approximately 10% of the CD4 co-receptor is associated with detergent-insoluble membrane microdomains, whilst the remaining 90% is in soluble membranes. Moreover, approximately 60% of the "insoluble CD4" is present in membrane microdomains containing GPI-anchored proteins and high glycolipid-dependent kinase activity, whereas the remaining 40% displays no association with GPI-anchored proteins and lacks glycolipid-associated kinase activity These results indicate that CD4 segregates at least into three different membrane microenvironments: 1) soluble membranes; 2) insoluble membrane microdomains containing GPI-anchored proteins; and 3) insoluble membrane microdomains devoid of GPI-anchored proteins. The level of CD4 in insoluble membranes was not modified upon triggering activation by T-cell receptor-crosslinking but detectable amounts of CD3 subunits were recruited into these specialized membranes under those conditions. The physical separation of CD4 into different membrane microenvironments raises the possibility of that some of the multiple functions of CD4 might segregate into distinct types of lipid microenvironment. The fact that components of T-cell receptor/CD3 complex were recruited into insoluble membranes upon stimulation is consistent with the CD4 present in this membrane fraction might participate in T-cell receptor-triggered activation events.

Animals↗
PubMed1999

MAL, an integral element of the apical sorting machinery, is an itinerant protein that cycles between the trans-Golgi network and the plasma membrane.

The MAL proteolipid is a nonglycosylated integral membrane protein found in glycolipid-enriched membrane microdomains. In polarized epithelial Madin-Darby canine kidney cells, MAL is necessary for normal apical transport and accurate sorting of the influenza virus hemagglutinin. MAL is thus part of the integral machinery for glycolipid-enriched membrane-mediated apical transport. At steady state, MAL is predominantly located in perinuclear vesicles that probably arise from the trans-Golgi network (TGN). To act on membrane traffic and to prevent their accumulation in the target compartment, integral membrane elements of the protein-sorting machinery should be itinerant proteins that cycle between the donor and target compartments. To establish whether MAL is an itinerant protein, we engineered the last extracellular loop of MAL by insertion of sequences containing the FLAG epitope or with sequences containing residues that became O-glycosylated within the cells or that displayed biotinylatable groups. The ectopic expression of these modified MAL proteins allowed us to investigate the surface expression of MAL and its movement through different compartments after internalization with the use of a combination of assays, including surface biotinylation, surface binding of anti-FLAG antibodies, neuraminidase sensitivity, and drug treatments. Immunofluorescence and flow cytometric analyses indicated that, in addition to its Golgi localization, MAL was also expressed on the cell surface, from which it was rapidly internalized. This retrieval implies transport through the endosomal pathway and requires endosomal acidification, because it can be inhibited by drugs such as chloroquine, monensin, and NH(4)Cl. Resialylation experiments of surface MAL treated with neuraminidase indicated that approximately 30% of the internalized MAL molecules were delivered to the TGN, probably to start a new cycle of cargo transport. Together, these observations suggest that, as predicted for integral membrane members of the late protein transport machinery, MAL is an itinerant protein cycling between the TGN and the plasma membrane.

Ammonium Chloride↗
PubMed1998

Thyroid hormone regulates the expression of the MAL proteolipid, a component of glycolipid-enriched membranes, in neonatal rat brain.

Detergent-insoluble glycosphingolipid-enriched membranes (DIGs) have been involved in the sorting and transport of specific proteins during oligodendrocyte maturation. The MAL (MAL, MVP17, VIP17) proteolipid, an integral membrane protein present in DIGs in mature oligodendrocytes, has been proposed as a component of the machinery for DIG-mediated transport in a restricted pattern of cell types including myelinating cells. We have previously shown that thyroid hormone regulates the expression of the myelin protein genes coordinately, and have suggested a major role for thyroid hormone in the control of oligodendrocytes generation. Here we show that the expression of the MAL gene is down-regulated by hypothyroidism and up-regulated by hyperthyroidism in myelinated regions of the brain. In contrast, adult-onset hypothyroidism has no effect on the steady-state levels of MAL mRNA. Taken together, our results show that MAL expression during oligodendrocyte maturation is modulated by thyroid hormone, suggesting that this hormone could play an important role in the myelin biogenesis during neonatal development.

Aging↗
PubMed1998

A short peptide motif at the carboxyl terminus is required for incorporation of the integral membrane MAL protein to glycolipid-enriched membranes.

The MAL (VIP17, MVP17) proteolipid, an integral membrane protein with specific residence in glycolipid-enriched membrane (GEM) microdomains, has been recently proposed as a component of the protein machinery for GEM vesiculation. In this work, we have searched the COOH terminus of MAL for sorting determinants responsible for targeting to GEMs. This has allowed the identification of the sequence Leu-Ile-Arg-Trp (LIRW) as necessary for the access of MAL to GEMs. This motif requires at least one additional amino acid at its COOH end for full effectiveness. The arginine within the LIRW motif is the most crucial residue for targeting to GEMs, tryptophan replacement affects targeting to a lesser extent, and the leucine-isoleucine pair tolerates substitution by valine, but not by alanine, without effect on targeting. Pulse-chase experiments indicate that the LIRW tetrapeptide is required for access to GEMs early after MAL biosynthesis. Interestingly, the loss of the capacity of the MAL protein to be incorporated into GEMs correlated with the loss of its response to brefeldin A treatment. This is the first identification of a juxtamembrane peptide motif required for incorporation of an integral membrane protein into GEMs.

Amino Acid Sequence↗
PubMed1998

An alternative domain determines nuclear localization in multifunctional protein 4.1.

Multiple protein 4.1 isoforms are originated by alternative pre-mRNA splicing, differential use of two translation initiation sites, and posttranslational modifications. The complexity of alternative splicing events suffered by the 4.1 pre-mRNA makes necessary the direct cloning of 4.1 full-coding cDNA sequences to ensure that the encoded 4.1 proteins are naturally occurring isoforms. We have approached this point by reverse transcription-polymerase chain reaction techniques using RNA from the nucleated human Molt-4 T-cell line as a starting template. Molecular cloning of 4.1 cDNAs using the second translation initiation codon has allowed us to identify two 4.1 isoforms, designated 4.1H and 4.1I, which are differentially targeted to the nucleus (4.1H) and the cytoplasm (4.1I). These two isoforms differ only in the inclusion (4.1H) or exclusion (4.1I) of 21 amino acids encoded by exon 16. A cluster of basic amino acids, KKKR, generated by joining of the sequences encoded by the constitutive exon 13 and the alternative exon 16, is necessary for the nuclear targeting of 4.1H, as demonstrated by site-directed mutagenesis analysis. Immunofluorescence microscopy and biochemical studies indicate that 4.1H belongs to the group of nuclear 4.1 proteins that are distributed diffusely throughout the nucleoplasm and that are extractable in 0.5% Triton X-100. This is the first demonstration of differential nuclear targeting by the presence of an alternative domain, among naturally occurring protein 4.1 isoforms.

Alternative Splicing↗
PubMed1998

MAL, a novel integral membrane protein of human T lymphocytes, associates with glycosylphosphatidylinositol-anchored proteins and Src-like tyrosine kinases.

A large fraction of glycosylphosphatidylinositol (GPI)-anchored proteins and Src-like kinases are confined to glycolipid-enriched membrane (GEM) microdomains. The particular membrane topology of GPI-anchored proteins has led to the postulation of the existence of integral membrane proteins linking extracellular stimuli with cytosolic machinery for endocytosis and signaling. The human MAL cDNA was identified during a search for novel genes differentially expressed during T cell development, and encodes a multispanning membrane protein displaying lipid-like properties. To address the biochemical characterization of endogenous MAL and to analyze its possible association with other proteins, we have generated a monoclonal antibody (mAb) specific to the MAL molecule. Using this mAb, we have identified MAL in GEM microdomains of both the HPB-ALL T cell line and human peripheral blood lymphocytes. Co-immunoprecipitation experiments with antibodies to the MAL molecule or to the GPI-anchored CD59 antigen indicated specific association of MAL with GPI-anchored proteins and Src-like tyrosine kinases. In addition, both MAL and the Src-like kinase Lck were identified in GEM obtained from an endosomal-enriched membrane fraction. These features of MAL closely match some of the properties expected for the hypothetical integral membrane linker proteins acting in specialized GEM-mediated functions.

Animals↗
PubMed1998

Expression of the MAL gene in the thyroid: the MAL proteolipid, a component of glycolipid-enriched membranes, is apically distributed in thyroid follicles.

The MAL proteolipid, an integral membrane protein expressed in T lymphocytes, polarized epithelial MDCK cells, and myelin-forming cells, has been identified as a component of internal glycolipid-enriched membrane (GEM) microdomains. On the basis of its ability to induce vesicle formation by ectopic expression, MAL has been recently proposed as a component of the machinery for GEM vesiculation. Taking into account the proposed role of GEMs in polarized transport, we have investigated the expression of the MAL gene in thyroid cells. Interestingly, MAL messenger RNA species were detected in the human thyroid, whereas they were undetectable in other endocrine glands tested. Moreover, epithelial FRT cells, a polarized rat cell line of thyroid origin, also expressed MAL transcripts. Immunohistochemical analysis of thyroid follicles, with a newly developed anti-MAL monoclonal antibody, indicates that MAL distribution is restricted to the apical zone of thyroid epithelial cells. Biochemical analyses, using FRT cells, indicate exclusive residence of MAL in GEM microdomains, and these analyses allowed the identification of MAL as a major protein component of the GEM fraction in this cell line. Our results are consistent with a role for MAL as a component of GEM microdomains in thyroid epithelial cells.

Amino Acid Sequence↗
PubMed1998

[Seroprevalence versus Parvovirus B19 in blood donors].

BACKGROUND: The aim of the study was to describe the seroprevalence against Parvovirus B19 in a random sample of blood donors in the Hospital Universitario de Salamanca. METHODS: We studied the presence of IgG and IgM antibodies against Parvovirus B19 in 136 sera from asymptomatic blood donors by enzyme immunoassay methods. RESULTS: From 136 samples tested, 88 (64.7%) had positive absorbance values for IgG. Forty eight samples (35.5%) were negative. IgM was negative in all cases. We did not find indeterminate results. DISCUSSION: Parvovirus primoinfection usually happens in the childhood. Thus, we can expect a high percentage of general population to have antibodies against Parvovirus B19. Anti-Parvovirus B19 antibodies prevalence in blood donors was 64.7%. This failure is similar to data reported before (65%). Clinical importance of these viruses in currently related with hemathopoyesis diseases and with the possible role in theratogenesis. The presence of IgG seems to give protection except in some chronic infections recently described.

Antibodies, Viral↗
PubMed1997

Multiple sorting signals determine apical localization of a nonglycosylated integral membrane protein.

In polarized cells, newly synthesized proteins are sorted in the trans-Golgi network and from there delivered to either the apical or basolateral membranes. Madin-Darby canine kidney (MDCK) cells have been widely used as a model system to study sorting determinants to the apical and basolateral surfaces. Whereas sorting signals for basolateral transmembrane proteins seem to reside in their cytoplasmic domains, apical determinants appear to reside in the N-glycans of secretory proteins or in the glycolipid tails of glycosylphosphatidylinositol-linked proteins. We show in this study that a surface-expressed form of CD3-epsilon, a nonglycosylated type I membrane protein, is exclusively targeted to the apical membrane in MDCK cells by a glycolipid-independent transport pathway. Deletion of the cytoplasmic tail does not affect its distribution, whereas deletion of the transmembrane domain results in secretion from both surfaces although still predominantly through the apical membrane. The transmembrane domain of CD3-epsilon appended to rat growth hormone, a secretory protein that lacks apical and basolateral determinants, promotes basolateral localization of the chimeric protein. However, a growth hormone chimera containing both the transmembrane and cytoplasmic domains of CD3-epsilon resulted in localization to the apical and basolateral membranes. These results suggest there are multiple determinants in CD3-epsilon that affect its distribution in polarized MDCK cells. Whereas the transmembrane domain contains a basolateral determinant, the ectodomain and the cytoplasmic domain contain apical determinants.

Animals↗
PubMed1997

Recombinant expression of the MAL proteolipid, a component of glycolipid-enriched membrane microdomains, induces the formation of vesicular structures in insect cells.

The MAL proteolipid has been identified as a component of glycolipid-enriched membrane microdomains resistant to detergent solubilization in epithelial Madin-Darby canine cells, as well as in T lymphocytes and in myelin-forming cells. To study the function of the MAL proteolipid we have ectopically expressed a tagged form of MAL in both mammalian and insect cellular backgrounds. Immunofluorescence analysis in transiently transfected COS-7 cells showed the presence of MAL in large vesicular structures, and biochemical analysis identified MAL in the fraction of membranes resistant to Triton X-100 solubilization. Electron microscopic analysis showed that the expression of MAL in Sf21 cells morphologically resulted in the intracellular accumulation of large vesicles with a diameter from 200 to greater than 700 nm that were absent in uninfected or control infected cultures. Thus, ectopic expression of MAL in this heterologous expression system was sufficient to drive the formation of vesicles with a size similar to that of the vesicles detected in mammalian cells. These vesicles were clearly different from the caveolae-like vesicles induced by caveolin expression, as evidenced by co-infection experiments using a recombinant caveolin baculovirus. Taken together, these results suggest that the MAL proteolipid might play a role as a component of the machinery of vesiculation of glycolipid-enriched membranes.

Animals↗
PubMed1997

Caveolin and MAL, two protein components of internal detergent-insoluble membranes, are in distinct lipid microenvironments in MDCK cells.

The MAL proteolipid and caveolin have been identified as components of internal detergent-insoluble membrane microdomains enriched in glycolipids and cholesterol. We have addressed the study of the glycolipid-enriched membranes in cells expressing endogenously only either MAL (Jurkat T cells) or caveolin (epithelial A498 cells) and in polarized MDCK cells which express both proteins simultaneously. Subcellular fractionation by centrifugation to equilibrium in sucrose gradients of Triton X-100 cell extracts from Jurkat and A498 cells revealed that MAL and caveolin are incorporated in detergent-insoluble buoyant membranes independently of the expression of each other and indicated the existence in these cells of insoluble membrane microdomains with either MAL or caveolin. Immunofluorescence analysis in MDCK cells indicated that both MAL and caveolin were located in the Golgi region, whereas caveolin was found in addition at the cell surface. Biochemical analysis in these cells revealed the existence of distinct membrane microenvironments differentially susceptible to detergent solubilization containing either internal MAL or internal plus surface caveolin. The observed heterogeneity within the internal glycolipid-enriched membrane fraction suggests the existence of distinct specialized lipid microenvironments in MDCK cells.

Animals↗