Regina Pohlmann

The sortilin cytoplasmic tail conveys Golgi–endosome transport and binds the VHS domain of the GGA2 sorting protein

Sortilin belongs to a growing family of multiligand type‐1 receptors with homology to the yeast receptor Vps10p. Based on structural features and sortilins intracellular predominance, we have proposed it to be a sorting receptor for ligands in the synthetic pathway as well as on the cell membrane. To test this hypothesis we examine here the cellular trafficking of chimeric receptors containing constructs of the sortilin tail. We report that sorting signals conforming to YXXΦ and dileucine motifs mediate rapid endocytosis of sortilin chimeras, which subsequently travel to the trans‐Golgi network, showing little or no recycling. Furthermore, we found that cation‐independent mannose 6‐phosphate receptor (MPR300)–sortilin chimeras, expressed in mannose 6‐phosphate receptor knockout cells, were almost as efficient as MPR300 itself for transport of newly synthesized β‐hexosaminidase and β‐glucuronidase to lysosomes, and established that the sortilin tail contains potent signals for Golgi–endosome sorting. Finally, we provide evidence suggesting that sortilin is the first example of a mammalian receptor targeted by the recently described GGA family of cytosolic sorting proteins, which condition the Vps10p‐mediated sorting of yeast carboxypeptidase Y.

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.

[46] Enzymic diagnosis of the genetic mucopolysaccharide storage disorders

Publisher Summary This chapter presents procedure for enzymic diagnosis of genetic mucopolysaccharide storage disorders particularly two enzyme defects underlying the Morquio syndrome. For the diagnosis of the rare disorders, fibroblasts are the most convenient enzyme source because of the possibility of performing extensive investigations and because of the relatively high activity found in normal cells. However, enzymic diagnosis can be performed also on leukocytes. α-glucosaminide N-acetyltransferase can be measured in cultured fibroblasts or amniotic fluid cells, leukocytes, and tissues, which is based on the principle that states the substrate, a trisaccharide with the structure O- (α-D-2-amino-2-deoxyglucopyranosyl)-(1 →4)-O-(β-D-glucopyranosyluronicacid)-(1→4)-2,5-anhydro-D-[ 3 H]mannitol, prepared from heparin, can be N-acetylated by acetyl-CoA:α-glucosaminide N-acetyltransferase in the presence of acetyl-CoA. The product bearing an N-acetylated glucosamine residue at the nonreducing terminal, can be hydrolyzed by α-N acetylglucosaminidase to N-acetylglucosamine and a radioactive disaccharide, which can be further split by β-glucuronidase. The positively charged substrate is separated from the neutral or negatively charged products by passage over a cation-exchange resin.

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.

Sorting by the Cytoplasmic Domain of the Amyloid Precursor Protein Binding Receptor SorLA

ABSTRACT SorLA/LR11 (250 kDa) is the largest and most composite member of the Vps10p-domain receptors, a family of type 1 proteins preferentially expressed in neuronal tissue. SorLA binds several ligands, including neurotensin, platelet-derived growth factor-bb, and lipoprotein lipase, and via complex-formation with the amyloid precursor protein it downregulates generation of Alzheimers disease-associated Aβ-peptide. The receptor is mainly located in vesicles, suggesting a function in protein sorting and transport. Here we examined SorLAs trafficking using full-length and chimeric receptors and find that its cytoplasmic tail mediates efficient Golgi body-endosome transport, as well as AP-2 complex-dependent endocytosis. Functional sorting sites were mapped to an acidic cluster-dileucine-like motif and to a GGA binding site in the C terminus. Experiments in permanently or transiently AP-1 μ1-chain-deficient cells established that the AP-1 adaptor complex is essential to SorLAs transport between Golgi membranes and endosomes. Our results further implicate the GGA proteins in SorLA trafficking and provide evidence that SNX1 and Vps35, as parts of the retromer complex or possibly in a separate context, are engaged in retraction of the receptor from endosomes.

Deficiency of UDP-N-acetylglucosamine: Lysosomal enzyme N-acetylglucosamine-1-phosphotransferase in organs of I-cell patients

Abstract A N-acetylglucosamine-1-phosphotransferase is involved in synthesis of a common phosphorylated recognition marker in lysosomal enzymes. Absence of this enzyme in liver, spleen, kidney and brain of two patients with I-cell disease is now reported. In these organs activities of lysosomal enzymes are close to normal. In contrast, in fibroblasts the absence of N-acetylglucosamine-1-phosphotransferase and of the common recognition marker are known to result in a severe intracellular deficiency of lysosomal enzymes. It is proposed that in certain organs the transport of lysosomal enzymes into lysosomes is mediated by alternative systems, which recognize structural features other than the phosphorylated recognition marker.

Human lysosomal acid phosphatase is transported as a transmembrane protein to lysosomes in transfected baby hamster kidney cells.

BHK cells transfected with human lysosomal acid phosphatase (LAP) cDNA (CT29) expressed 70‐fold higher enzyme activities of acid phosphatase than non‐transfected BHK cells. The CT29‐LAP was synthesized in BHK cells as a heterogeneously glycosylated precursor that was tightly membrane associated. Transfer to the trans‐Golgi was associated with a small increase in size (approximately 7 kd) and partial processing of the oligosaccharides to complex type structures. CT29‐LAP was transferred into lysosomes as shown by subcellular fractionation, immunofluorescence and immunoelectron microscopy. Lack of mannose‐6‐phosphate residues suggested that transport does not involve mannose‐6‐phosphate receptors. Part of the membrane‐associated CT29‐LAP was processed to a soluble form. The mechanism that converts CT29‐LAP into a soluble form was sensitive to NH4Cl, and reduced the size of the polypeptide by 7 kd. In vitro translation of CT29‐derived cRNA in the presence of microsomal membranes yielded a CT29‐LAP precursor that is protected from proteinase K except for a small peptide of approximately 2 kd. In combination with the sequence data available for LAP, these observations suggest that CT29‐LAP is synthesized and transported to lysosomes as a transmembrane protein. In the lysosomes, CT29‐LAP is released from the membrane by proteolytic cleavage, which removes a C‐terminal peptide including the transmembrane domain and the cytosolic tail of 18 amino acids.

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.

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.

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.