Biomedical subjects

R Pohlmann

Publications and source records attributed to R Pohlmann.

At least 19 recordsLinked to original sources

PubMed1995

The two mannose 6-phosphate receptors transport distinct complements of lysosomal proteins.

Mammalian cells express two different mannose 6-phosphate receptors (MPR 46 and MPR 300), which both mediate targeting of Man-6-P-containing lysosomal proteins to lysosomes. To assess the contribution of either and both MPRs to the transport of lysosomal proteins, fibroblasts were established from mouse embryos that were homozygous for disrupted alleles of either MPR 46 or MPR 300 or both MPRs. Fibroblasts missing both MPRs secreted most of the newly synthesized lysosomal proteins and were unable to maintain the catabolic function of lysosomes. The intracellular levels of lysosomal proteins decreased to < 20%, and undigested material accumulated in the lysosomal compartment. Fibroblasts lacking either MPR exhibited only a partial missorting and maintained, in general, half-normal to normal levels of lysosomal proteins. The same species of lysosomal proteins were found in secretions of double MPR-deficient fibroblasts as in secretions of single MPR-deficient fibroblasts, but at different ratios. This clearly indicates that neither MPR has an exclusive affinity for one or several lysosomal proteins. Furthermore, neither MPR can substitute in vivo for the loss of the other. It is proposed that the heterogeneity of the Man-6-P recognition marker within a lysosomal protein and among different lysosomal proteins has necessitated the evolution of two MPRs with complementary binding properties to ensure an efficient targeting of lysosomal proteins.

Animals↗

PubMed1994

Mistargeting of lysosomal enzymes in M(r) 46,000 mannose 6-phosphate receptor-deficient mice is compensated by carbohydrate-specific endocytotic receptors.

Targeted disruption of the M(r) 46,000 mannose 6-phosphate receptor (MPR 46) in mice is associated with normal levels of lysosomal enzymes in the circulation, while in MPR 46-deficient cells an increased secretion of lysosomal enzymes is apparent [Köster, A., Saftig, P., Matzner, U., von Figura, K., Peters, C. & Pohlmann, R. (1993) EMBO J. 12, 5219-5223]. This points to the existence of mechanisms that prevent or compensate for mistargeting of lysosomal enzymes in vivo. In the present study, we have injected inhibitors of three carbohydrate-specific endocytotic receptors into MPR 46-deficient and control mice. Inhibition of these receptors was associated with a pronounced increase of three lysosomal enzymes in the serum of MPR 46-deficient mice. These results clearly show that lysosomal enzymes are mistargeted in MPR 46-deficient mice and that carbohydrate-specific endocytotic receptors are part of the mechanisms that compensate for the mistargeting of lysosomal enzymes in MPR 46-deficient mice. Moreover, evidence was obtained that, also in control mice, the steady-state level of some lysosomal enzyme is controlled by these receptors.

Animals↗

PubMed1993

Targeted disruption of the M(r) 46,000 mannose 6-phosphate receptor gene in mice results in misrouting of lysosomal proteins.

Lysosomal enzymes containing mannose 6-phosphate recognition markers are sorted to lysosomes by mannose 6-phosphate receptors (MPRs). The physiological importance of this targeting mechanism is illustrated by I-cell disease, a fatal lysosomal storage disorder caused by the absence of mannose 6-phosphate residues in lysosomal enzymes. Most mammalian cells express two MPRs. Although the binding specificities, subcellular distribution and expression pattern of the two receptors can be differentiated, their coexpression is not understood. The larger of the two receptors with an M(r) of approximately 300,000 (MPR300), which also binds IGFII, appears to have a dominant role in lysosomal enzyme targeting, while the function of the smaller receptor with an M(r) of 46,000 (MPR46) is less clear. To investigate the in vivo function of the MPR46, we generated MPR46-deficient mice using gene targeting in embryonic stem cells. Reduced intracellular retention of newly synthesized lysosomal proteins in cells from MPR46 -/- mice demonstrated an essential sorting function of MPR46. The phenotype of MPR46 -/- mice was normal, indicating mechanisms that compensate the MPR46 deficiency in vivo.

Animals↗

PubMed1992

Localization of lysosomal acid phosphatase mRNA in mouse tissues.

We studied the expression of lysosomal acid phosphatase (LAP) in mouse by hybridizing Northern blots and tissue sections with the mouse LAP cDNA. Three mRNA species of 2.3, 3.2 and 5.2 KB were identified, which differ in the length of their 3' untranslated region (UTR). The 3.2 KB mRNA is expressed in equal amounts in all tissues and represents the major species in most tissues, whereas the amounts of the 2.3 and 5.2 KB species differ. In situ hybridization of different tissues of adult mice showed a uniform expression of LAP, as expected for a housekeeping gene, except in testis and brain. In testis we found an increase in the LAP mRNA level in spermatocytes. By Northern blot analysis of young mouse testis, this increase could be attributed to late pachytene primary spermatocytes or secondary spermatocytes. In brain tissue the neurons were predominantly labeled, especially the Purkinje and pyramidal cells, whereas glial cells expressed only low amounts of LAP mRNA. Very high LAP expression was also found in the epithelial cells of the choroid plexus. Analysis of LAP expression during mouse embryonic development between Days 9.5 and 17.5 revealed a prominent expression relative to other tissues in the neural tube from Day 9.5 to Day 13.5.

Acid Phosphatase↗

PubMed1992

Expression of the two mannose 6-phosphate receptors is spatially and temporally different during mouse embryogenesis.

Mammalian cells express two mannose 6-phosphate receptors, MPR46 and MPR300, both of which mediate the targeting of lysosomal enzymes to lysosomes. Additionally the receptors mediate the secretion (MPR46) and the endocytosis (MPR300) of lysosomal enzymes and the binding of IGFII (MPR300). We have analyzed the distribution of MPR46 and MPR300 transcripts during mouse embryogenesis by in situ hybridization. Up to day 15.5 of embryonic development we found a non-overlapping distribution of the transcripts for the two receptors. High expression of MPR46 was observed at sites of hemopoiesis and in the thymus while MPR300 was highly expressed in the cardiovascular system. Late in embryogenesis (day 17.5) a wide variety of tissues expressed the receptors, but still the expression pattern was almost non-overlapping. This unexpected complementary expression pattern points to specific functions of the two mannose 6-phosphate receptors during mouse embryogenesis.

Animals↗

PubMed1991

Mutational analysis of disulfide bridges in the Mr 46,000 mannose 6-phosphate receptor. Localization and role for ligand binding.

Formation of intramolecular disulfide bonds is a key step in the early maturation of newly synthesized Mr 46,000 mannose 6-phosphate receptors to acquire ligand-binding activity (Hille, A., Waheed, A., and von Figura, K. (1990) J. Cell Biol. 110, 963-972). The luminal domain of the receptor, which carries the ligand-binding site, contains 6 cysteine residues. We have analyzed the function of individual cysteine residues for the ligand-binding conformation by exchanging cysteine for glycine. In each case, the replacement of cysteine resulted in a complete loss of binding activity, indicating that all 6 luminal cysteine residues are required for the ligand-binding conformation. The cysteine mutants displayed a greatly reduced immunoreactivity, decreased stability, and a blocked or delayed transport to the trans Golgi. The glycosylation pattern allowed the distinguishing of three phenotypes, each of which was represented by one pair of cysteine mutants. Based on the assumption that replacement of either of the 2 cysteine residues forming a disulfide bond results in an identical phenotype, we postulate that disulfide bonds are formed between Cys-32 and Cys-78 and between Cys-132 and Cys-167, as well as between Cys-145 and Cys-179. This assumption was supported by the observation that the simultaneous exchange of the 2 cysteine residues of a putative pair resulted in the same phenotypes as the single exchange of either of the 2 cysteine residues.

Animals↗

PubMed1991

Isolation and analysis of the human 46-kDa mannose 6-phosphate receptor gene.

From a genomic library in EMBL 3, two overlapping clones for the human 46-kDa mannose 6-phosphate receptor (MPR46) were isolated, which span the entire coding sequence. The human MPR46 gene is distributed over 12 kb and is divided into seven exons (110-1573 bp). All the intron/exon borders agree with the consensus sequences of splice junctions. Exon 1 codes for a 5' untranslated sequence. The ATG initiation codon begins with the second nucleotide in exon 2. A signal sequence of 26 amino acid residues is followed by the extracytoplasmic (luminal) domain, which extends to exon 5. The transmembrane domain of the receptor spans exons 5 and 6 and the cytoplasmic domain is encoded by exons 6 and 7. The latter domain also codes for an extended 3' untranslated sequence. The transcription-initiation site was defined by primer extension. The sequence upstream of the cap site has strong promoter activity and contains structural elements characteristic of promoters found in housekeeping genes. No correlation between the genomic organization and known protein domains of the MPR46 was apparent. Moreover, the sequence of about 150 amino acids within the luminal domain of MPR46, which is homologous to the 15 repeats that constitute the luminal domain of the 300-kDa mannose 6-phosphate receptor (MPR300), does not correlate with intron/exon borders. MPR46 and MPR300 have therefore diverged from a common ancestral gene before introduction of the present intron sequences.

Amino Acid Sequence↗

PubMed1991

Glycosylation of the Mr 46,000 mannose 6-phosphate receptor. Effect on ligand binding, stability, and conformation.

Using site-directed mutagenesis the N-glycosylation sites of the Mr 46,000 mannose 6-phosphate receptor (MPR 46) were identified as asparagine residues 57, 83, 107, and 113. The two outer asparagines carry high mannose-type and the two inner asparagines carry complex-type oligosaccharides. The glycosylation mutants were analyzed for stability, binding activity, and subcellular distribution. Replacing asparagine 57, 83, or 107 by threonine decreased only the stability of the receptor. Replacing asparagine 113 by threonine decreased the stability and binding activity. Deletion of three or all four N-glycosylation sites led in addition to an accumulation of the mutant receptors in endoplasmic reticulum-like structures. Nonglycosylated MPR 46 synthesized in the presence of tunicamycin, thus preserving the asparagine residues, had a normal stability and high affinity binding. The decreased stability and binding activity of the receptor mutants is therefore due to the exchange of asparagine residues rather than to the loss of N-linked oligosaccharides. The nonglycosylated receptor, however, displayed a decreased conformational stability after solubilization as a single cycle of freezing and thawing reduced the binding activity to one-third of the control. Simultaneously, the receptor lost its quaternary structure. It is concluded from these results that the N-glycosylation of the receptor is required for the stability of a high affinity conformation, but not for the binding itself or the intracellular stability.

Cell Compartmentation↗

PubMed1991

Mr 46,000 mannose 6-phosphate receptor. The role of histidine and arginine residues for binding of ligand.

The chemical modification of histidine and arginine residues results in a loss of binding of the Mr 46,000 mannose 6-phosphate receptor (MPR 46) to a phosphomannan affinity matrix (Stein, M., Meyer, J. E., Hasilik, A., and von Figura, K. (1987) Biol. Chem. Hoppe-Seyler 368, 927-936). Reversal of the modification or presence of mannose 6-phosphate during the modification partially restores or protects the binding activity, indicating that histidine and arginine residues contribute to the mannose 6-phosphate binding site. The 5 histidine and 8 arginine residues within the luminal domain of MPR 46, which contains the ligand binding site, were exchanged by site-directed mutagenesis. Only the conservative replacement of His-131 and Arg-137 by serine and lysine, respectively, results in a loss of binding activity without affecting other properties of the receptor such as the presence of intramolecular disulfide bonds, immunoreactivity, processing of N-linked oligosaccharides, formation of dimers, intracellular distribution, and surface expression. Conservative replacement of other histidine and arginine residues did not affect the binding activity. Nonconservative replacement of several arginine residues reduced binding activity and immunoreactivity, indicating that the loss of a positive charge at these positions alters the folding of MPR 46. We conclude from these results that His-131 and Arg-137 are essential for binding of ligands by MPR 46.

Arginine↗

PubMed1991

Molecular cloning of the mouse 46-kDa mannose 6-phosphate receptor (MPR 46).

A cDNA clone for the mouse 46-kDa mannose 6-phosphate receptor (MPR 46) was isolated from an embryonic mouse cDNA library. Its single open reading frame codes for a protein of 278 residues. It shows an over-all amino-acid identity of 93% with the human receptor. Nine non-conservative amino-acid exchanges are found in the luminal domain, one non-conservative exchange of hydrophobic amino acids is in the transmembrane domain, while the cytoplasmic receptor tails are identical. All five potential N-glycosylation sites are conserved as well as amino acids that are important for ligand binding (Arg 137 and His 131) and disulfide pairing (Cys 32 and 78, Cys 132 and Cys 167, Cys 145 and Cys 179). The absolute identity in the cytoplasmic MPR 46 tail suggests the importance of this amino-acid sequence for the intracellular routing of the MPR 46.

Amino Acid Sequence↗

PubMed1991

Molecular cloning of the mouse lysosomal acid phosphatase.

The mouse cDNA for lysosomal acid phosphatase was cloned. The deduced amino-acid sequence shows 89 and 96% identity with that of the human and rat enzyme, respectively. Namely all residues known to be important for the structure, catalytic activity and transport of lysosomal acid phosphatase are conserved among the three species.

Acid Phosphatase↗

PubMed1990

Phylogenetic conservation of arylsulfatases. cDNA cloning and expression of human arylsulfatase B.

A 2.2-kilobase cDNA clone for human arylsulfatase B (ASB) and several genomic clones were isolated and sequenced. The deduced amino acid sequence of 533 amino acids contains a 41-amino acid N-terminal signal peptide and a mature polypeptide of 492 amino acid residues. Overexpression of ASB in transfected baby hamster kidney (BHK) cells resulted in up to 68-fold higher ASB activity than in untransfected BHK cells. Pulse-chase labeling showed that ASB was synthesized and secreted as a 64-kDa precursor and processed to a 47-kDa mature form in BHK cells. The 47-kDa ASB form was located in dense lysosomes. Transport of ASB to the lysosomes was accomplished in a mannose 6-phosphate receptor-dependent manner. The ASB cDNA clone hybridizes to 4.8-, 2.5-, and 1.8-kilobase species of RNA from human fibroblasts. The same pattern was observed in RNA from fibroblasts of three Maroteaux-Lamy patients who were deficient in ASB activity, as well as in RNA from fibroblasts of three patients with multiple sulfatase deficiency, in which all known sulfatases were markedly diminished. Deduced amino acid sequences of human arylsulfatase A, human ASB, human steroid sulfatase, human glucosamine-6-sulfatase, and an arylsulfatase from sea urchin showed a substantial degree of similarity suggesting that they arose from a common ancestral gene and are members of an arylsulfatase gene family.

Amino Acid Sequence↗

PubMed1990

Gene expression and prostate specificity of human prostatic acid phosphatase (PAP): evaluation by RNA blot analyses.

A fragment of a complementary DNA (cDNA) clone for human prostatic acid phosphatase (PAP) (EC 3.1.3.2.) was used to study the expression of corresponding mRNA in human tissues. The specificity of its expression in benign prostatic hyperplasia (BPH) and prostatic carcinoma tissues were indicated in RNA blot analyses. The PAPcDNA probe did not recognize any specific mRNAs in RNAs extracted from human liver cancer, lung cancer, pancreatic cancer, placenta, breast cancer cells (MCF-7), mononuclear blood cells or acute promyelocytic leukemia cells (HL-60), according to Northern blot analysis. mRNA for PAP was detected in the androgen-dependent human prostatic cancer cell line LNCaP, but not in the androgen-insensitive human prostatic cancer cell line PC-3. In contrast, lysosomal acid phosphatase (LAP) mRNA was detected in both of these human prostatic cancer cell lines. Our findings indicate a high specificity for the PAP gene in prostatic tissue. The mean abundance for the PAPmRNA expression was 0.26 for prostatic carcinoma samples (n = 11) and 0.46 for BPH samples (n = 8) according to slot-blot analysis. The differences observed between the different categories of prostatic tissue in PAPmRNA abundances call for additional studies on regulation of its expression.

Acid Phosphatase↗

PubMed1990

Targeting of a lysosomal membrane protein: a tyrosine-containing endocytosis signal in the cytoplasmic tail of lysosomal acid phosphatase is necessary and sufficient for targeting to lysosomes.

Lysosomal acid phosphatase (LAP) is synthesized as a transmembrane protein with a short carboxy-terminal cytoplasmic tail of 19 amino acids, and processed to a soluble protein after transport to lysosomes. Deletion of the membrane spanning domain and the cytoplasmic tail converts LAP to a secretory protein, while deletion of the cytoplasmic tail as well as substitution of tyrosine 413 within the cytoplasmic tail against phenylalanine causes accumulation at the cell surface. A chimeric polypeptide, in which the cytoplasmic tail of LAP was fused to the ectoplasmic and transmembrane domain of hemagglutinin is rapidly internalized and tyrosine 413 of the LAP tail is essential for internalization of the fusion protein. A chimeric polypeptide, in which the membrane spanning domain and cytoplasmic tail of LAP are fused to the ectoplasmic domain of the Mr 46 kd mannose 6-phosphate receptor, is rapidly transported to lysosomes, whereas wild type receptor is not transported to lysosomes. We conclude that a tyrosine containing endocytosis signal in the cytoplasmic tail of LAP is necessary and sufficient for targeting to lysosomes.

Acid Phosphatase↗

PubMed1990

Mannose 6-phosphate receptor dependent secretion of lysosomal enzymes.

BHK and mouse L cells transfected with the cDNA for the human 46 kd mannose 6-phosphate receptor (MPR 46) secrete excessive amounts of newly synthesized mannose 6-phosphate containing polypeptides. The secretion is dependent on the amount, the recycling and the affinity for ligands of MPR 46. Incubation of transfected cells with antibodies blocking the binding site of MPR 46 reduces the secretion, and cotransfection with the cDNA for the human 300 kd mannose 6-phosphate (MPR 300) restores it to normal values. These results indicate that the two mannose 6-phosphate receptors compete for binding of newly synthesized ligands. In contrast to ligands bound to MPR 300, those bound to the MPR 46 are transported to and released at a site, e.g. early endosomes or plasma membrane, from where they can exit into the medium. Since antibodies blocking the binding site of MPR 46 reduce secretion also in non-transfected BHK and mouse L cells, at least part of the basal secretion of M6P-containing polypeptides is mediated by the endogenous MPR 46.

Animals↗

PubMed1989

Structure of the human lysosomal acid phosphatase gene.

We have isolated a 12-kb genomic clone, which encodes human lysosomal acid phosphatase (LAP), a lysosomal membrane glycoprotein. The human LAP gene has a size of about 9 kb and contains 11 exons (83-947 bp in size). The signal sequence and the first eight amino acids of the LAP protein are encoded by exon 1, the remaining luminal domain by exons 2-10 and the transmembrane and cytoplasmic domains, as well as the 3'-untranslated region, by exon 11. The sequence of the LAP gene confirmed the sequence deduced from the cDNA clone except for nucleotide 1917 in the 3'-untranslated region, where T is changed to C. The 5'-flanking sequence shows promoter activity, as analysed by coupling to bacterial chloramphenicol acetyltransferase. S1-nuclease-protection and primer-extension analysis demonstrate transcription initiation at multiple sites clustering within 23 bp upstream of the translation-initiation codon. Sequences characteristic for promoter regions like TATA-box and CAAT-box sequences could not be identified at typical positions. The absence of these sequences, the high GC content (63.5%), two GC boxes and a region complying with the properties of a CpG island, indicate that LAP is a housekeeping gene.

Acid Phosphatase↗

PubMed1989

Synthesis of a truncated Mr 46,000 mannose 6-phosphate receptor that is secreted and retains ligand binding.

The Mr 46,000 mannose 6-phosphate receptor is an integral membrane protein with its ligand-binding site in the ectoplasmic domain. By site-directed mutagenesis, a stop codon was introduced in the receptor cDNA at the border between the ectoplasmic and membrane-spanning domain. The truncated receptor was expressed in three different systems, Xenopus oocytes, COS cells and BHK-21 cells. In all three systems the truncated receptor behaved as a soluble protein. In oocytes only small amounts of the truncated receptor were secreted within 48 h after synthesis. Accumulation of endoglucosaminidase H-sensitive forms of the truncated receptor in oocytes suggested that exit from the endoplasmic reticulum was slowed down. In COS and BHK-21 cells, the truncated receptor was secreted and, as for wild-type receptor, most of the N-linked oligosaccharides were processed to complex forms. Both the intracellularly-retained (oocytes) and the secreted (COS and BHK-21 cells) truncated receptors bound to phosphomannan-Sepharose in a mannose-6-phosphate-dependent manner. Using chemical cross-linking, the truncated receptor was shown to be secreted as a homodimer.

Animals↗

PubMed1989

Cloning and expression of human arylsulfatase A.

A full length cDNA for human arylsulfatase A was cloned and sequenced. The predicted amino acid sequence comprises 507 residues. A putative signal peptide of 18 residues is followed by the NH2-terminal sequence of placental arylsulfatase A. One of the arylsulfatase A peptides ends 3 residues ahead of the predicted COOH terminus. This indicates that proteolytic processing of arylsulfatase A is confined to the cleavage of the signal peptide. The predicted sequence contains three potential N-glycosylation sites, two of which are likely to be utilized. The sequence shows no homology to any of the known sequences of lysosomal enzymes but a 35% identity to human steroid sulfatase. Transfection of monkey and baby hamster kidney cells resulted in an up to 200-fold increase of the arylsulfatase A activity. The arylsulfatase A was located in lysosome-like structures and transported to dense lysosomes in a mannose 6-phosphate receptor-dependent manner. The arylsulfatase A cDNA hybridizes to 2.0- and 3.9-kilobase species in RNA from human fibroblasts and human liver. RNA species of similar size were detected in metachromatic leukodystrophy fibroblasts of two patients, in which synthesis of arylsulfatase A polypeptides was either detectable or absent.

Amino Acid Sequence↗