Andrej Hasilik

Intracellular receptor sorting during endocytosis: Comparative immunoelectron microscopy of multiple receptors in rat liver

Using double-label quantitative immunoelectron microscopy on ultrathin cryosections of rat liver, we have compared the endocytotic pathways of the receptors for asialoglycoprotein (ASGP-R), mannose-6-phosphate ligands (MP-R), and polymeric IgA (IgA-R). All three were found within the Golgi complex, along the entire plasma membrane, in coated pits and vesicles, and within a compartment of uncoupling of receptors and ligand ( CURL ). The receptors occurred randomly at the cell surface, in coated pits and vesicles. Within CURL tubules ASGP-R and MP-R were colocalized , but IgA-R and ASGP-R displayed dramatic microheterogeneity. Thus, in addition to its role in uncoupling and sorting recycling receptor from ligand, CURL serves as a compartment to segregate recycling receptor (e.g. ASGP-R) from receptor involved in transcytosis (e.g. IgA-R).

Enzymatic phosphorylation of lysosomal enzymes in the presence of UDP-N-acetylglucosamine. Absence of the activity in l-cell fibroblasts

Abstract Recent finding of α-N-acetylglucosamine(1)phospho(6)mannose diesters in lysosomal enzymes suggested that formation of mannose 6-phosphate residues involves transfer of N-acetylglucosamine 1-phosphate to mannose. Using dephosphorylated β-hexosaminidase as acceptor and [β- 32 P]UDP-N-acetylglucosamine as donor for the phosphate group, phosphorylation of β-hexosaminidase by microsomes from rat liver, human placenta and human skin fibroblasts was achieved. The reaction was not affected by tunicamycin. Acid hydrolysis released mannose 6-[ 32 P]phosphate from the phosphorylated β-hexosaminidase. Our results suggest that lysosomal enzymes are phosphorylated by transfer of N-acetylglucosamine 1-phosphate from UDP-N-acetylglucosamine. The transferase activity was deficient in fibroblasts from patients affected with l-cell disease. This deficiency is proposed to be the primary enzyme defect in l-cell disease.

Integral and Associated Lysosomal Membrane Proteins

We searched for novel proteins in lysosomal membranes, tentatively participating in molecular transport across the membrane and/or in interactions with other compartments. In membranes purified from placental lysosomes, we identified 58 proteins, known to reside at least partially in the lysosomal membrane. These included 17 polypeptides comprising or associated with the vacuolar adenosine triphosphatase. We report on additional 86 proteins that were significantly enriched in the lysosomal membrane fraction. Among these, 12 novel proteins of unknown functions were found. Three were orthologues of rat proteins that have been identified in tritosomes by Bagshaw RD et al. (A proteomic analysis of lysosomal integral membrane proteins reveals the diverse composition of the organelle. Mol Cell Proteomics 2005;4:133–143). Here, the proteins encoded by LOC201931 (FLJ38482) and LOC51622 (C7orf28A) were expressed with an appended fluorescent tag in HeLa cells and found to be present in lysosomal organelles. Among the lysosomally enriched proteins, also 16 enzymes and transporters were detected that had not been assigned to lysosomal membranes previously. Finally, our results identified a particular set of proteins with known functions in signaling and targeting to be at least partially associated with lysosomes.

Selective inactivity of TGF-β/decorin complexes

Previous studies had shown that binding of TGF‐β to the small proteoglycan decorin results in its inactivation. Indeed, in osteosarcoma cells the addition of decorin prevented the TGF‐β1‐mediated up‐regulation of biglycan synthesis. However, the down‐regulation of proteoglycan‐100 remained unaltered. Even in the presence of a 100,000‐fold molar excess of decorin, TGF‐β1 was fully active in U937 monocytes with respect to the inhibition of cell proliferation. There was no inhibition of the TGF‐β‐mediated stimulation of the retraction of fibroblast‐populated collagen lattices. Thus, the formation of TGF‐β/decorin complexes leads to the neutralization of distinct effects only.

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.

The proteome of lysosomes

Lysosomes are organelles of eukaryotic cells that are critically involved in the degradation of macromolecules mainly delivered by endocytosis and autophagocytosis. Degradation is achieved by more than 60 hydrolases sequestered by a single phospholipid bilayer. The lysosomal membrane facilitates interaction and fusion with other compartments and harbours transport proteins catalysing the export of catabolites, thereby allowing their recycling. Lysosomal proteins have been addressed in various proteomic studies that are compared in this review regarding the source of material, the organelle/protein purification scheme, the proteomic methodology applied and the proteins identified. Distinguishing true constituents of an organelle from copurifying contaminants is a central issue in subcellular proteomics, with additional implications for lysosomes as being the site of degradation of many cellular and extracellular proteins. Although many of the lysosomal hydrolases were identified by classical biochemical approaches, the knowledge about the protein composition of the lysosomal membrane has remained fragmentary for a long time. Using proteomics many novel lysosomal candidate proteins have been discovered and it can be expected that their functional characterisation will help to understand functions of lysosomes at a molecular level that have been characterised only phenomenologically so far and to generally deepen our understanding of this indispensable organelle.

Mr 46,000 mannose 6-phosphate specific receptor: its role in targeting of lysosomal enzymes.

Antibodies that block the ligand binding site of the cation‐dependent mannose 6‐phosphate specific receptor (Mr 46,000 MPR) were used to probe the function of the receptor in transport of lysosomal enzymes. Addition of the antibodies to the medium of Morris hepatoma 7777 cells, which express only the Mr 46,000 MPR, resulted in a decreased intracellular retention and increased secretion of newly synthesized lysosomal enzymes. In fibroblasts and HepG2 cells that express the cation‐independent mannose 6‐phosphate specific receptor (Mr 215,000 MPR) in addition to the Mr 46,000 MPR, antibodies against the Mr 46,000 MPR inhibited the intracellular retention of newly synthesized lysosomal enzymes only when added to the medium together with antibodies against the Mr 215,000 MPR. Morris hepatoma (M.H.) 7777 did not endocytose lysosomal enzymes, while U937 monocytes, which express both types of MPR, internalized lysosomal enzymes. The uptake was inhibited by antibodies against the Mr 215,000 MPR, but not by antibodies against the Mr 46,000 MPR. These observations suggest that Mr 46,000 MPR mediates transport of endogenous but not endocytosis of exogenous lysosomal enzymes. Internalization of receptor antibodies indicated that the failure to mediate endocytosis of lysosomal enzymes is due to an inability of surface Mr 46,000 MPR to bind ligands rather than its exclusion from the plasma membrane or from internalization.

Mannose 6-phosphate/insulin-like growth factor II receptor: Distinct binding sites for mannose 6-phosphate and insulin-like growth factor II

Pentamannosyl phosphate substituted bovine serum albumin (PMP-BSA) and insulin like growth factor II (IGF II) bind specifically to immobilized mannose 6-phosphate/insulin like growth factor II receptor. An excess of IGF II inhibited binding of PMP-BSA by less than or equal to 20%, and an excess of PMP-BSA inhibited binding of IGF II by less than or equal to 10%. Polyclonal antibodies against the receptor purified from human liver inhibited preferentially the binding of PMP-BSA, and a monocloncal antibody 2C2 inhibited only the binding of IGF II to the receptor. Similar results were obtained for binding of PMP-BSA and IGF II to human skin fibroblasts. These results suggest that the binding sites for mannose 6-phosphate and IGF II reside in different portions of the receptor.

Purified recombinant human prosaposin forms oligomers that bind procathepsin D and affect its autoactivation

Before delivery to endosomes, portions of proCD (procathepsin D) and proSAP (prosaposin) are assembled into complexes. We demonstrate that such complexes are also present in secretions of cultured cells. To study the formation and properties of the complexes, we purified proCD and proSAP from culture media of Spodoptera frugiperda cells that were infected with baculoviruses bearing the respective cDNAs. The biological activity of proCD was demonstrated by its pH-dependent autoactivation to pseudocathepsin D and that of proSAP was demonstrated by feeding to saposin-deficient cultured cells that corrected the storage of radioactive glycolipids. In gel filtration, proSAP behaved as an oligomer and proCD as a monomer. ProSAP altered the elution of proCD such that the latter was shifted into proSAP-containing fractions. ProSAP did not change the elution of mature cathepsin D. Using surface plasmon resonance and an immobilized biotinylated proCD, binding of proSAP was demonstrated under neutral and weakly acidic conditions. At pH 6.8, specific binding appeared to involve more than one binding site on a proSAP oligomer. The dissociation of the first site was characterized by a K(D1) of 5.8+/-2.9x10(-8) M(-1) (calculated for the monomer). ProSAP stimulated the autoactivation of proCD and also the activity of pseudocathepsin D. Concomitant with the activation, proSAP behaved as a substrate yielding tri- and disaposins and smaller fragments. Our results demonstrate that proSAP forms oligomers that are capable of binding proCD spontaneously and independent of the mammalian type N-glycosylation but not capable of binding mature cathepsin D. In addition to binding proSAP, proCD behaves as an autoactivable and processing enzyme and its binding partner as an activator and substrate.

Carbohydrate-free carboxypeptidase Y is transferred into the lysosome-like yeast vacuole

Abstract Carboxypeptidase Y, localized in the lysosome-like yeast vacuole, has been metabolically labeled with [2- 3 H]mannose. After immunoprecipitation the carbohydrate moieties were released by treatment with endo-β-N-acetyl-glucosaminidase H and separated by paper electrophoresis. Evidence for the presence of phospho-monoester and -diester groups in the molecule has been obtained. In the latter phosphate links C-1 of mannose or of mannosyl 1,3-mannose to C-6 of a mannose residue within a larger oligomannose moiety. In the presence of tunicamycin yeast cells synthesize a carbohydrate-free carboxypeptidase Y, which could be traced after metabolic labeling with [ 14 C]-phenylalanine. The carbohydrate-free enzyme was segregated into the vacuoles to the same extent as the intact glycoprotein.