Stuart Kornfeld

Mannose 6-phosphate receptors: new twists in the tale

The two mannose 6-phosphate (M6P) receptors were identified because of their ability to bind M6P-containing soluble acid hydrolases in the Golgi and transport them to the endosomal–lysosomal system. During the past decade, we have started to understand the structural features of these receptors that allow them to do this job, and how the receptors themselves are sorted as they pass through various membrane-bound compartments. But trafficking of acid hydrolases is only part of the story. Evidence is emerging that one of the receptors can regulate cell growth and motility, and that it functions as a tumour suppressor.

The mannose 6-phosphate receptor and the biogenesis of lysosomes

Localization of the 215 kd mannose 6-phosphate receptor (MPR) was studied in normal rat kidney cells. Low levels of receptor were detected in the trans Golgi network, Golgi stack, plasma membrane, and peripheral endosomes. The bulk of the receptor was localized to an acidic, reticular-vesicular structure adjacent to the Golgi complex. The structure also labeled with antibodies to lysosomal enzymes and a lysosomal membrane glycoprotein (lgp120). While lysosome-like, this structure is not a typical lysosome that is devoid of MPRs. The endocytic marker alpha 2 macroglobulin-gold entered the structure at 37 degrees C, but not at 20 degrees C. With prolonged chase, most of the marker was transported from the structure into lysosomes. We propose that the MPR/lgp-enriched structure is a specialized endosome (prelysosome) that serves as an intermediate compartment into which endocytic vesicles discharge their contents, and where lysosomal enzymes are released from the MPR and packaged along with newly synthesized lysosomal glycoproteins into lysosomes.

Trafficking of lysosomal enzymes.

The targeting of lysosomal enzymes from their site of synthesis in the rough endoplasmic reticulum (RER) to their final destination in lysosomes is directed by a series of protein and carbohydrate recognition signals on the enzymes. Lysosomal enzymes, along with secretory and plasma membrane proteins, contain aminoterminal signal sequences that direct the vectorial discharge of the nascent proteins into the lumen of the RER. The three classes of proteins also share a common peptide signal for asparagine glycosylation. The next signal is unique to lysosomal enzymes and permits their high‐affinity binding to a specific phosphotransferase that catalyzes the formation of the mannose 6‐phosphate recognition marker. This carbohydrate determinant allows binding to specific receptors that translocate the lysosomal enzymes from the Golgi complex to an acidified prelysosomal compartment. There the lysosomal enzymes are discharged for final packaging into lysosomes. Two distinct mannose 6‐phosphate receptors have been identified, and cDNAs encoding their entire sequences have been cloned. An analysis of the deduced amino acid sequences of the receptors shows that each is composed of four structural domains: a signal sequence, an extracytoplasmic amino‐terminal domain, a hydrophobic membrane‐spanning region, and a cytoplasmic domain. The entire extracytoplasmic region of the small receptor is homologous to the 15 repeating domains that constitute the extracytoplasmic portion of the large receptor.— Kornfeld, S. Trafficking of lysosomal enzymes. FASEB J. 1: 462‐468; 1987.

The trans-Golgi network: a late secretory sorting station

Proteins synthesized on membrane-bound ribosomes are transported through the Golgi apparatus and, on reaching the trans-Golgi network, are sorted for delivery to various cellular destinations. Sorting involves the assembly of cytosol-oriented coat structures which preferentially package cargo into vesicular transport intermediates. Recent studies have shed new light on both the molecular machinery involved and the complexity of the sorting processes.

Mutation of the COG complex subunit gene COG7 causes a lethal congenital disorder.

The congenital disorders of glycosylation (CDG) are characterized by defects in N-linked glycan biosynthesis that result from mutations in genes encoding proteins directly involved in the glycosylation pathway. Here we describe two siblings with a fatal form of CDG caused by a mutation in the gene encoding COG-7, a subunit of the conserved oligomeric Golgi (COG) complex. The mutation impairs integrity of the COG complex and alters Golgi trafficking, resulting in disruption of multiple glycosylation pathways. These cases represent a new type of CDG in which the molecular defect lies in a protein that affects the trafficking and function of the glycosylation machinery.

Structure of Glycoproteins and Their Oligosaccharide Units

The presence of oligosaccharide chains covalently attached to the peptide backbone is the feature that distinguishes glycoproteins from other proteins and accounts for some of their characteristic physical and chemical properties. Glycoproteins occur in fungi, green plants, viruses, bacteria, and in higher animal cells where they serve a variety of functions. Connective tissue glycoproteins, such as the collagens and proteoglycans of various animal species, are structural elements as are the cell wall glycoproteins of yeasts and green plants. The submaxillary mucins and the glycoproteins in the mucous secretions of the gastrointestinal tract, which consist of numerous oligosaccharide chains attached at closely spaced intervals to a peptide backbone, serve as lubricants and protective agents. The body fluids of vertebrates are rich in glycoproteins secreted from various glands and organs. Constituents of blood plasma which are glycoproteins include the transport proteins transferrin, ceruloplasmin, and transcobalamin I as well as the immunoglobulins, all the known clotting factors, and many of the components of complement. Follicle-stimulating hormone, luteinizing hormone, and thyroid-stimulating hormone (secreted by the pituitary) and chorionic gonadotropin are all glycoproteins as are the enzymes ribonuclease and deoxyribonuclease (secreted by the pancreas) and α-amylase (secreted by the salivary glands). Fungi secrete a number of glycoprotein enzymes, for example, Taka-amylase and invertase. Another group of glycoproteins are those which occur as integral components of cell membranes in a variety of species. Enveloped viruses contain surface glycoproteins that are involved in the attachment of the virus to its host, and in eukaryotic cells the histocompatibility antigens are membrane glycoproteins. There is a growing body of evidence to suggest that cell surface glycoproteins are involved in a number of physiologically important functions such as cell-cell interaction, adhesion of cells to substratum, and migration of cells to particular organs, for example, the “homing” of lymphocytes to the spleen and the metastasis of tumor cells to preferred sites.

Symbol Nomenclature for Graphical Representations of Glycans

Author(s): Varki, Ajit; Cummings, Richard D; Aebi, Markus; Packer, Nicole H; Seeberger, Peter H; Esko, Jeffrey D; Stanley, Pamela; Hart, Gerald; Darvill, Alan; Kinoshita, Taroh; Prestegard, James J; Schnaar, Ronald L; Freeze, Hudson H; Marth, Jamey D; Bertozzi, Carolyn R; Etzler, Marilynn E; Frank, Martin; Vliegenthart, Johannes Fg; Lutteke, Thomas; Perez, Serge; Bolton, Evan; Rudd, Pauline; Paulson, James; Kanehisa, Minoru; Toukach, Philip; Aoki-Kinoshita, Kiyoko F; Dell, Anne; Narimatsu, Hisashi; York, William; Taniguchi, Naoyuki; Kornfeld, Stuart

Mutations in the cytoplasmic domain of the 275 kd mannose 6-phosphate receptor differentially alter lysosomal enzyme sorting and endocytosis

The cation-independent mannose 6-phosphate receptor (Cl-MPR) sorts newly synthesized lysosomal enzymes in the Golgi and endocytoses extracellular lysosomal enzymes. To determine the role of the 163 amino acid cytoplasmic domain of the Cl-MPR in these functions, receptor-deficient mouse L cells were transfected with normal bovine Cl-MPR cDNA or cDNAs mutated in the cytoplasmic domain. The normal Cl-MPR functioned in sorting and endocytosis. Mutant receptors with 40 and 89 residues deleted from the carboxyl terminus of the cytoplasmic tail functioned normally in endocytosis, but were partially impaired in sorting. Mutant receptors with larger deletions leaving only 7 and 20 residues of the cytoplasmic tail were defective in endocytosis and sorting. A mutant receptor containing alanine instead of tyrosine residues at positions 24 and 26 was defective in endocytosis, and partially impaired in sorting. Receptors deficient in endocytosis accumulated at the cell surface. These results indicate that the cytoplasmic domain of the Cl-MPR contains different signals for rapid endocytosis and efficient lysosomal enzyme sorting.

The iminosugar isofagomine increases the activity of N370S mutant acid β-glucosidase in Gaucher fibroblasts by several mechanisms

Gaucher disease is a lysosomal storage disorder caused by deficiency in lysosomal acid β-glucosidase (GlcCerase), the enzyme responsible for the catabolism of glucosylceramide. One of the most prevalent disease-causing mutations, N370S, results in an enzyme with lower catalytic activity and impaired exit from the endoplasmic reticulum. Here, we report that the iminosugar isofagomine (IFG), an active-site inhibitor, increases GlcCerase activity 3.0 ± 0.6-fold in N370S fibroblasts by several mechanisms. A major effect of IFG is to facilitate the folding and transport of newly synthesized GlcCerase in the endoplasmic reticulum, thereby increasing the lysosomal pool of the enzyme. In addition, N370S GlcCerase synthesized in the presence of IFG exhibits a shift in pH optimum from 6.4 to 5.2 and altered sensitivity to SDS. Although IFG fully inhibits GlcCerase in the lysosome in an in situ assay, washout of the drug leads to partial recovery of GlcCerase activity within 4 h and full recovery by 24 h. These findings provide support for the possible use of active-site inhibitors in the treatment of some forms of Gaucher disease.

The rapid induction by phytohemagglutinin of increased alpha-aminoisobutyric acid uptake by lymphocytes.

The effect of phytohemagglutinin (PHA) on the ability of human lymphocytes to transport the nonutilizable amino acid, alpha-aminoisobutyric acid (AIB) has been studied. PHA binds rapidly to plasma membrane receptor sites with half maximal binding requiring approximately 7.5 min. During the first 30 min after PHA addition to lymphocytes no change was detected in AIB transport, but then a linear increase in the initial rate of AIB transport occurred over the next 9 hr. Subsequently, the rate of AIB transport stabilized at a level 6-7 times greater than that found in control lymphocytes. The change in membrane function developed even when de novo protein synthesis was inhibited by 85-90% with puromycin or cycloheximide. However, the PHA effect did not occur when the lymphocytes were maintained at 4 degrees C. Studies of the kinetics of AIB uptake by control and PHA-treated lymphocytes demonstrated that PHA increases the V(max) of AIB uptake by 6-7-fold (0.7 mmumole AIB per 10(6) lymphocytes/15 min versus 0.1 mmumole per 10(6) lymphocytes/15 min) without affecting the Km (Michaelis constant) of the transport system (2mM in both cases).When fetuin was added to lymphocyte cultures to remove bound PHA, the PHA-induced increase in the rate of AIB uptake was arrested at the rate achieved during the time of prior incubation with PHA. This level of AIB transport persisted for at least 3 hr after 80% of the PHA was removed from the cell membrane. These data demonstrate that PHA rapidly induces a change in a lymphocyte cell membrane transport function, and that the continued presence of PHA on the cell membrane is required for the full stimulatory effect to be reached. The data do not distinguish between a direct action of PHA upon the lymphocyte membrane or the possibility that PHA slowly enters into the cell where it then exerts its effect.