K von Figura

Mice deficient for the lysosomal proteinase cathepsin D exhibit progressive atrophy of the intestinal mucosa and profound destruction of lymphoid cells.

Mice deficient for the major lysosomal aspartic proteinase cathepsin D, generated by gene targeting, develop normally during the first 2 weeks, stop thriving in the third week and die in a state of anorexia at day 26 +/− 1. An atrophy of the ileal mucosa first observed in the third week progresses towards widespread intestinal necroses accompanied by thromboemboli. Thymus and spleen undergo massive destruction with fulminant loss of T and B cells. Lysosomal bulk proteolysis is maintained. These results suggest, that vital functions of cathepsin D are exerted by limited proteolysis of proteins regulating cell growth and/or tissue homeostasis, while its contribution to bulk proteolysis in lysosomes appears to be non‐critical.

A di-leucine-based motif in the cytoplasmic tail of LIMP-II and tyrosinase mediates selective binding of AP-3.

Among the various coats involved in vesicular transport, the clathrin associated coats that contain the adaptor complexes AP‐1 and AP‐2 are the most extensively characterized. The function of the recently described adaptor complex AP‐3, which is similar to AP‐1 and AP‐2 in protein composition but does not associate with clathrin, is not known. By monitoring surface plasmon resonance we observed that AP‐3 is able to interact with the tail of the lysosomal integral membrane protein LIMP‐II and that this binding depends on a DEXXXLI sequence in the LIMP‐II tail. Furthermore, AP‐3 bound to the cytoplasmic tail of the melanosome‐associated protein tyrosinase which contains a related EEXXXLL sequence. The tails of LIMP‐II and tyrosinase either did not interact, or only interacted poorly, with AP‐1 or AP‐2. In contrast, the cytoplasmic tails of other membrane proteins containing di‐leucine and/or tyrosine‐based sorting signals did not bind AP‐3, but AP‐1 and/or AP‐2. This points to a function of AP‐3 in intracellular sorting to lysosomes and melanosomes of a subset of cargo proteins via di‐leucine‐based sorting motifs.

Lysosomal acid phosphatase is transported to lysosomes via the cell surface.

Lysosomal acid phosphatase (LAP) is transported as a transmembrane protein to dense lysosomes. The pathway of LAP to lysosomes includes the passage through the plasma membrane. LAP is transported from the trans‐Golgi to the cell surface with a half‐time of less than 10 min. Cell surface LAP is rapidly internalized. Most of the internalized LAP is transported back to the cell surface. On average, each LAP molecule cycles greater than 15 times between the cell surface and the endosomes before it is transferred to dense lysosomes. At equilibrium approximately 4 times more LAP precursor is present in endosomes than at the cell surface. Exposing cells to reduced temperature or weak bases such as NH4Cl, chloroquine and primaquine decreases the steady‐state concentration of LAP at the cell surface. The recycling pathway is operative at greater than or equal to 20 degrees C and does not include passage of the Golgi/trans‐Golgi network. LAP is transferred with a half‐time of 5‐6 h from the plasma membrane/endosome pool to dense lysosomes, from where it does not recycle to the endosome/plasma membrane pool at a measurable rate.

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.

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.

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.

Carbohydrate-deficient glycoprotein syndromes become congenital disorders of glycosylation: an updated nomenclature for CDG. First International Workshop on CDGS

During the last few years, progress in identifying the molecular defects of the carbohydrate-deficient glycoprotein syndromes has been very rapid. Up to this date, six different gene defects have been elucidated. The plethora of defects that will eventually be identified makes it indispensable to use a simple and straightforward nomenclature for this group of diseases.A group of specialists in this field met for a round-table discussion at the “First International Workshop on CDGS” in Leuven, Belgium, November 12–13, 1999, and came up with the following recommendations.1. CDG stands for “Congenital Disorders of Glycosylation”.2. The disorders are divided into groups, based on the biochemical pathway affected: group I refers to defects in the initial steps of N-linked protein glycosylation. These deficiencies affect the assembly of dolichylpyrophosphate linked oligosaccharide and/or its transfer to asparagine residues on the nascent polypeptides; group II refers to defects in the processing of protein-bound glycans or the addition or other glycans to the protein. This grouping no longer refers directly to the isoelectric focusing pattern of serum transferrins or other serum glycoproteins.3. CDG types are assigned to one of the groups and will be numbered consecutively as they are identified: Ia, Ib,...[emsp4 ], IIa, IIb,...[emsp4 ], etc. The currently distinguished types are: CDG-Ia (PMM2[emsp4 ]), CDG-Ib (MPI[emsp4 ]), CDG-Ic (ALG6[emsp4 ]), CDG-Id (ALG3[emsp4 ]), CDG-Ie (DPM1), CDG-IIa (MGAT2[emsp4 ]).4. No new designations will be made unless the genetic defect is established. Untyped cases are considered “x” cases (CDG-x) until the genetic defect is known.

The cytoplasmic tail of lysosomal acid phosphatase contains overlapping but distinct signals for basolateral sorting and rapid internalization in polarized MDCK cells.

Lysosomal acid phosphatase (LAP) is synthesized as a type I membrane glycoprotein and targeted to lysosomes via the plasma membrane. Its cytoplasmic tail harbours a tyrosine‐containing signal for rapid internalization. Expression in Madine‐Darby canine kidney cells results in direct sorting to the basolateral cell surface, rapid endocytosis and delivery to lysosomes. In contrast, a deletion mutant lacking the cytoplasmic tail is delivered to the apical plasma membrane where it accumulates before it is slowly internalized. A chimeric protein, in which the cytoplasmic tail of LAP is fused to the extracytoplasmic and transmembrane domain of the apically sorted haemagglutinin, is sorted to the basolateral plasma membrane. A series of truncation and substitution mutants in the cytoplasmic tail was constructed and comparison of their polarized sorting and internalization revealed that the determinants for basolateral sorting and rapid internalization reside in the same segment of the cytoplasmic tail. The cytoplasmic factors decoding these signals, however, tolerate distinct mutations indicating that different receptors are involved in sorting at the trans‐Golgi network and at the plasma membrane.

Amyloidogenic Processing of Human Amyloid Precursor Protein in Hippocampal Neurons Devoid of Cathepsin D

βA4-Amyloid peptide, the main component of the amyloid plaques in the brain of Alzheimers disease patients is produced from amyloid precursor protein (APP) by proteolytical processing. Several lines of evidence suggest a direct role for cathepsin D, the major endosomal/lysosomal aspartic endopeptidase, in βA4-amyloid peptide generation. Here we tested this hypothesis using primary cultures of hippocampal neurons derived from cathepsin D-deficient (knock out) mice and expressing wild-type human APP and two clinical APP variants via recombinant Semliki Forest virus. We demonstrate APP secretory processing, production of carboxyl-terminal amyloid fragments, and secretion of the βA4-amyloid peptide in the complete absence of cathepsin D. The results rule out cathepsin D as a critical component of α-, β-, or γ-secretase and therefore as a primary target for drugs aimed at decreasing the βA4-amyloid peptide burden in Alzheimers disease.

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.