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E Ikonen

Publications and source records attributed to E Ikonen.

At least 55 records · Page 3Linked to original sources
PubMed1994

Basolateral protein transport in streptolysin O-permeabilized MDCK cells.

We have reconstituted polarized protein transport in streptolysin O-permeabilized MDCK cells from the TGN to the basolateral surface and to the apical surface. These transport steps are dependent on temperature, energy and exogenously supplied cytosol. Using this in vitro system we show that a whole tail peptide (WT peptide) corresponding to the cytoplasmic tail of a basolaterally sorted protein, the vesicular stomatitis virus glycoprotein (VSV G) inhibits the TGN to basolateral transport but does not affect any other transport step. Inhibition of VSV G transport to basolateral surface by WT peptide did not result in missorting of the protein to the apical surface. Mutation of the single tyrosine residue in the WT peptide reduced its inhibitory potency four- to fivefold. These results suggest that the VSV G tail physically interacts with a component of the sorting machinery. Using a cross-linking approach, we have identified proteins that associate with the cytoplasmic tail domain of VSV G. One of these polypeptides, Tin-2 (Tail interacting protein-2), associates with VSV G in the TGN, the site of protein sorting, but not in the ER nor at the cell surface. Tin-2 does not associate with apically targeted hemagglutinin. WT peptide that inhibited the basolateral transport of VSV G also inhibited the association of Tin-2 with VSV G. Together, these properties make Tin-2 a candidate basolateral sorter. The results demonstrate the usefulness of the SLO-permeabilized cell system in dissecting the sorting machinery.

Amino Acid Sequence↗
PubMed1994

Dissection of the molecular consequences of a double mutation causing a human lysosomal disease.

Aspartylglucosaminidase (AGA) is a lysosomal enzyme, the deficiency in which leads to human storage disease aspartylglucosaminuria (AGU). AGUFin is the most common AGU mutation in the world and is found in 98% of AGU alleles in Finland, where the population displays enrichment of the disease allele. The AGUFin allele actually contains a double mutation, both individual mutations resulting in amino acid substitutions: Arg-161-->Gln and Cys-163-->Ser. The separate consequences of these two amino acid substitutions for the intracellular processing of the AGA polypeptides were analyzed using a stable expression of mutant polypeptides in Chinese hamster ovary (CHO) cells. The synthesized polypeptides were monitored by metabolic labeling, followed by immunoprecipitation, immunofluorescence, and immunoelectron microscopy. The Arg-161-->Gln substitution did not affect the intracellular processing or transport of AGA and the fully active enzyme was correctly targeted to lysosomes. The Cys-163-->Ser substitution prevented the early proteolytic cleavage required for the activation of the precursor AGA polypeptide and the inactive enzyme was accumulated in the endoplasmic reticulum (ER). The precursors of the translation products of the AGUFin double mutant and the Cys-163-->Ser mutant were also observed in the culture medium. When cells expressing the normal AGA or AGUFin double mutation were treated with DTT to prevent the formation of disulfide bonds, both normal and mutated AGA polypeptides remained in the inactive precursor form and were not secreted into the medium. These results indicate that correct initial folding is essential for the proteolytic activation of AGA.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Semliki Forest virus as a tool for protein expression in cultured rat hippocampal neurons.

We use the Semliki Forest Virus (SFV) as a tool for protein expression in primary cultures of rat hippocampal neurons. These cells develop in vitro into polarized neurons that can be infected with recombinant SFV with an efficiency of 60 - 80%. SFV-driven protein expression is detectable within 3-4 hours postinfection, at which time the newly-synthesized proteins are mainly present in the cell soma. By 6 to 8 hours postinfection foreign proteins are detectable in the neurites. Protein expression can continue for up to 48 hours. However, after 8 - 10 hours infected neurons start to suffer from cytopathic effects as evidenced by a change in morphology and detachment from the coverslip. The infection does not seem to affect the polarized distribution of proteins. Upon overexpression of rab8, a somatodendritic distribution is observed, similar to that of the endogenous protein. Therefore, the SFV expression system is suitable for short-term expression of proteins and can be used successfully to study the polarized distribution of heterologous proteins expressed in cultured hippocampal neurons.

Animals↗
PubMed1993

Transcytosis of the polymeric immunoglobulin receptor in cultured hippocampal neurons.

BACKGROUND: A wide variety of proteins are transported across epithelial cells by vesicular carriers. This process, transcytosis, is used to generate cell surface polarity and to transport macromolecules between the luminal and serosal sides of the epithelial layer. The polymeric immunoglobulin receptor is a well-characterized transcytotic molecule in epithelia. It binds to its ligand, polymeric immunoglobulin, at the basolateral surface, and the receptor-ligand complex is transcytosed to the apical surface, where the ligand is released. Our previous studies have shown that hippocampal neurons may employ mechanisms similar to those of epithelial cells to sort proteins to two plasma membrane domains. The machinery used for axonal delivery recognizes proteins that are targeted apically in epithelia, whereas basolaterally destined proteins are delivered to the dendrites. It has not been clear, however, whether transcytosis occurs in neurons. RESULTS: We report expression of the polymeric immunoglobulin receptor in cultured hippocampal neurons, using a Semliki Forest Virus expression system, and show by immunofluorescence microscopy that the newly synthesized receptor is targeted from the Golgi complex predominantly to the dendrites - only about 20% of the infected neurons display axonal immunofluorescence. Addition of ligand leads to significant redistribution of the receptor to the axons, shown by an approximately three-fold increase in axonal immunoreactivity with the anti-receptor antibodies. CONCLUSIONS: Our results suggest that a transcytotic route, analogous to that in epithelia, exists in neurons, where it transports proteins from the somatodendritic to the axonal domain. Cultured neurons expressing the polymeric immunoglobulin receptor offer an experimental system that should be useful for further characterization of this novel neuronal pathway at the molecular and functional level.

Journal Article↗
PubMed1993

Lysosomal aspartylglucosaminidase is processed to the active subunit complex in the endoplasmic reticulum.

Aspartylglucosaminidase (AGA) is a lysosomal enzyme, the deficiency of which leads to a human storage disease, aspartylglucosaminuria (AGU). Although numerous mutations have been identified in AGU patients, elucidation of the molecular pathogenesis of the disease has been hampered by the missing information on the cellular events resulting in the maturation and activation of the enzyme. Here we used the expression of in vitro mutagenized constructs of the AGA cDNA to define three specific proteolytic trimming steps resulting in mature AGA. Removal of the signal peptide is immediately followed by proteolytic cleavage of the precursor into two subunits and results in biologically active enzyme already in the endoplasmic reticulum. This early activation has not previously been described for lysosomal enzymes. The subsequent lysosomal trimming does not influence the enzymatic activity of AGA. It consists only of a single proteolytic cleavage which removes 10 amino acids from the C-terminal end of the larger subunit, in contrast to the multistep lysosomal processing observed in several other hydrolases.

Amino Acid Sequence↗
PubMed1993

Dissection of the molecular pathology of aspartylglucosaminuria provides the basis for DNA diagnostics and future therapeutic interventions.

Aspartylglucosaminuria (AGU) is exceptional among lysosomal storage diseases since it represents the only known amidase deficiency in man, being caused by an inadequate function of aspartylglucosaminidase (AGA, E.C. 3.5.1.26.). This amidase is essential in one of the final steps in the ordered breakdown of glycoproteins since it cleaves Asn from the residual N-acetylglucosamines (for reviews see 1, 2). The deficiency of the enzyme activity results in the typical lysosomal accumulation of the abnormal degradation products (mainly aspartylglucosamine, 2-acetamido-1-beta-L-aspartamido-1,2-dideoxyglucose) in patients' cells and tissues. The diagnosis of AGU has so far been based on the detection of abnormal metabolites in urine and decreased enzyme activity in the cultured fibroblasts or isolated lymphocytes. Prenatal diagnosis has been possible by demonstrating the deficient enzyme activity of amniocytes or chorion villus biopsies. Identification of carriers has been difficult and unreliable due to the high individual variation in AGA activity and prerequisite for isolated blood lymphocytes. During the past few years we have purified the human enzyme into homogeneity, isolated the full length cDNA and characterized the majority of AGU mutations in this cDNA. This work facilitated the development of a reliable DNA diagnostic test suitable also for large scale carrier screening. The molecular pathology of the most common AGU mutation was unravelled, this being a prerequisite for the oncoming developments for therapy. Although AGU is a relatively rare disease, characterization of the AGU mutations and their cellular consequences have revealed highly interesting new phenomena in the biosynthesis of this lysosomal enzyme, some of which carry general biological significance.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylglucosamine↗
PubMed1992

Terminal deletion of chromosome 4p (4p16.3) shows a breakpoint between loci linked to Huntington disease.

A 15-year-old boy with a terminal deletion of the short arm of chromosome 4 is described. The patient has a mild clinical phenotype that is incompatible with Wolf-Hirschhorn syndrome. Careful neurological examination including CT scan did not show any signs of Huntington disease. The chromosomal breakpoint was analyzed by means of polymorphic DNA probes localized close to the tentative Huntington (HD) locus. The breakage has occurred between D4S43 and D4S90 loci and thus deletes part of the chromosomal candidate regions for the HD locus.

Abnormalities, Multiple↗
PubMed1992

Deletion of the 3'-untranslated region of aspartylglucosaminidase mRNA results in a lysosomal accumulation disease.

Aspartylglucosaminuria (AGU) is a lysosomal storage disease due to mutations in the aspartylglucosaminidase (AGA) gene. The deficient enzyme activity in patients' cells blocks one of the final steps in the degradation of N-linked glycoproteins. All the AGU mutations identified so far affect the coding region of the AGA gene. Here we report a homozygous 876-base pair deletion, which removes the 3'-noncoding area but leaves the coding region of the AGA mRNA intact. This deletion does not prevent transcription termination or polyadenylation of the patient's truncated mRNA, and the steady state level of the mRNA is comparable with the control. However, the quantity of AGA polypeptide chains in the patient's fibroblasts is negligible. This suggests that the deletion interferes with the translational efficiency in vivo and provides a unique model to pursue the biological significance of untranslated regions of human mRNAs.

Aspartylglucosylaminase↗
PubMed1992

Mutations causing aspartylglucosaminuria (AGU): a lysosomal accumulation disease.

This article provides a review of the mutations reported so far in the lysosomal storage disease aspartylglucosaminuria (AGU). The clinical symptoms, biochemical findings, and diagnostic possibilities of the disease are introduced. The prevalence and biological consequences of the found mutations are then described, as well as the availability of a new rapid DNA test suitable for carrier screening. This test will be especially applicable in the genetically isolated Finnish population, where the carrier frequency of AGU was found to be as high as 1:36. Finally, future prospects dealing with the foreseeable therapeutic interventions of the disease are discussed.

Acetylglucosamine↗
PubMed1992

Huntington disease in Finland: a molecular and genealogical study.

Huntington disease (HD) is found at exceptionally low frequency in the Finnish population. In this population, linkage disequilibrium was earlier established with markers from the D4S10 and D4S43 loci. We now report a continuation to the restriction fragment length polymorphism haplotype analysis, in combination with a genealogical study of all the Finnish HD families. When the HD pedigrees were systematically traced to the 18th century, only one consanguinity was found, and a high percentage (28%) of the families had foreign ancestors. The majority of the Finnish ancestors were localized to border regions or trade centers of the country following the old postal routes. The observed high risk haplotypes formed with markers from the D4S10 and D4S43 loci were evenly distributed among the HD families in different geographical locations. Consequently, the HD gene(s) has most probably arrived in Finland on several occasions via foreign immigrants during the last few centuries.

Chromosomes, Human, Pair 4↗
PubMed1992

Convenient and quantitative determination of the frequency of a mutant allele using solid-phase minisequencing: application to aspartylglucosaminuria in Finland.

Aspartylglucosaminuria (AGU) is a recessively inherited lysosomal disease caused by inadequate aspartylglucosaminidase (AGA) activity. The disease is prevalent in the genetically isolated Finnish population. We have used a new method, solid-phase minisequencing, to determine the frequency of two missense mutations in the AGA gene in this population. In samples from 70% of the Finnish AGU families, we found that the two nucleotide changes were always associated, and they were identified in 98% of the AGU alleles analyzed. Thus, the high prevalence of AGU in the Finnish population is the consequence of a founder effect of one ancient mutation. The identification of asymptomatic carriers by the minisequencing test proved to be unequivocal. The method also allowed quantification of a mutated nucleotide sequence present in less than 1% of a sample. The frequency of AGU carriers in this population was 1/36 when estimated by quantifying the mutated AGU allele in a pooled leukocyte sample from 1350 normal Finnish individuals.

Acetylglucosamine↗