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S Kornfeld

Publications and source records attributed to S Kornfeld.

At least 91 records · Page 5Linked to original sources
PubMed1987

The interaction of phosphorylated oligosaccharides and lysosomal enzymes with bovine liver cation-dependent mannose 6-phosphate receptor.

We have analyzed the interaction of phosphorylated oligosaccharides and lysosomal enzymes with immobilized bovine liver cation-dependent mannose-6-P receptor. Oligosaccharides with phosphomonoesters were the only species that interacted with the receptor, and molecules with two phosphomonoesters showed the best binding. Lysosomal enzymes with several oligosaccharides containing only one phosphomonoester had a higher affinity for the receptor than did the isolated oligosaccharides, indicating the possible importance of multivalent interactions between weakly binding ligands and the receptor. The binding of a mixture of phosphorylated lysosomal enzymes to the cation-dependent Man-6-P receptor was markedly influenced by pH. At pH 6.3, almost all of the lysosomal enzymes bound to the receptor; whereas at pH 7.0-7.5, approximately one-third of the material passed through the column, one-third interacted weakly, and one-third bound tightly. The distribution of individual lysosomal enzyme activities was similar to that of the total material. The species of phosphorylated oligosaccharides present on the lysosomal enzymes which interacted poorly with the receptor were similar to those found on the tightly bound material and included species of oligosaccharides with two phosphomonoester groups. Isolated oligosaccharides of this type bound to the receptor over the entire pH range tested. These findings indicate that at neutral pH the phosphorylated oligosaccharides on some lysosomal enzyme molecules are oriented in a manner which makes them inaccessible to the binding site of the cation-dependent Man-6-P receptor. Since the same enzymes bind to the cation-independent Man-6-P receptor at neutral pH, at least a portion of the phosphomannosyl residues must be exposed. We conclude that small variations in the pH of the Golgi compartment where lysosomal enzymes bind to the receptors could potentially modulate the extent of binding to the two receptors.

Animals↗
PubMed1987

Cloning of the bovine 215-kDa cation-independent mannose 6-phosphate receptor.

Four overlapping cDNA clones encoding a partial sequence of the cation-independent 215-kDa mannose 6-phosphate receptor have been identified by screening a fetal calf liver cDNA library with oligonucleotide probes. RNA hybridization analysis showed that the length of the mRNA is approximately 9.5 kilobases. Sequence analysis demonstrated that the clones consist of 4647 contiguous nucleotides and contain an open reading frame coding for a polypeptide of 1461 amino acids, which we estimate represents greater than 75% of the primary structure of the receptor. The deduced amino acid sequence indicates that the receptor has a carboxyl-terminal cytoplasmic domain of 163 amino acids that is rich in acidic residues, a 23-amino acid transmembrane segment, and an extracellular domain containing at least eight homologous repeats of approximately 145 amino acids. One of the repeats contains an additional 43-residue segment that is similar to the type II repeat of fibronectin. Each repeat contains a highly conserved 13-amino acid unit bordered by cysteine residues that may be functionally important.

Amino Acid Sequence↗
PubMed1987

Expression of human cathepsin D in Xenopus oocytes: phosphorylation and intracellular targeting.

We have obtained expression of a cDNA clone for human cathepsin D in Xenopus laevis oocytes. Biosynthetic studies with [35S]methionine labeling demonstrated that most of the cathepsin D remained intracellular and underwent proteolytic cleavage, converting a precursor of Mr 47,000 D to a mature form of Mr 39,000 D with processing intermediates of Mr 43,000-41,000 D. greater than 90% of the cathepsin D synthesized by oocytes bound to a mannose 6-phosphate (Man-6-P) receptor affinity column, indicating the presence of phosphomannosyl residues. An analysis of [2-3H]mannose-labeled oligosaccharides directly demonstrated phosphomannosyl residues on cathepsin D. Sucrose-gradient fractionation, performed to define the membranous compartments that cathepsin D traversed during its biosynthesis, demonstrated that cathepsin D is targeted to a subpopulation of yolk platelets, the oocyte equivalent of a lysosome. Xenopus oocytes were able to endocytose lysosomal enzymes from the medium and this uptake was inhibited by Man-6-P, thus demonstrating the presence of Man-6-P receptors in these cells. Therefore, the entire Man-6-P dependent pathway for targeting of lysosomal enzymes is present in the oocytes. Xenopus oocytes should be a useful system for examining signals responsible for the specific targeting of lysosomal enzymes to lysosomes.

Animals↗
PubMed1987

Renin, a secretory glycoprotein, acquires phosphomannosyl residues.

Renin is an aspartyl protease which is highly homologous to the lysosomal aspartyl protease cathepsin D. During its biosynthesis, cathepsin D acquires phosphomannosyl residues that enable it to bind to the mannose 6-phosphate (Man-6-P) receptor and to be targeted to lysosomes. The phosphorylation of lysosomal enzymes by UDP-GlcNAc:lysosomal enzyme N-acetylglucosaminylphosphotransferase (phosphotransferase) occurs by recognition of a protein domain that is thought to be present only on lysosomal enzymes. In order to determine whether renin, being structurally similar to cathepsin D, also acquires phosphomannosyl residues, human renin was expressed from cloned DNA in Xenopus oocytes and a mouse L cell line and its biosynthesis and posttranslational modifications were characterized. In Xenopus oocytes, the majority of the renin remained intracellular and underwent a proteolytic cleavage which removed the propiece. Most of the renin synthesized by oocytes was able to bind to a Man-6-P receptor affinity column (53%, 57%, and 90%, in different experiments), indicating the presence of phosphomannosyl residues. In the L cells, the majority of the renin was secreted but 5-6% of the renin molecules contained phosphomannosyl residues as demonstrated by binding of [35S]methionine-labeled renin to the Man-6-P receptor as well as direct analysis of [2-3H]mannose-labeled oligosaccharides. Although the level of renin phosphorylation differed greatly between the two cell types examined, these results demonstrate that renin is recognized by the phosphotransferase and suggest that renin contains at least part of the lysosomal protein recognition domain.

Animals↗
PubMed1987

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 amino-terminal 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.

Animals↗
PubMed1986

Glycoprotein phosphorylation in simple eucaryotic organisms. Identification of UDP-GlcNAc:glycoprotein N-acetylglucosamine-1-phosphotransferase activity and analysis of substrate specificity.

UDP-N-acetylglucosamine:glycoprotein N-acetylglucosamine-1-phosphotransferase activity has been identified in both Acanthamoeba castellani and Dictyostelium discoideum. Each of these activities exhibits a different in vitro specificity toward various purified glycoproteins. The N-acetylglucosaminyl-phosphotransferase of A. castellani is very similar to the mammalian enzyme in that it phosphorylates the lysosomal enzymes cathepsin D and uteroferrin much more efficiently than nonlysosomal glycoproteins and appears to recognize a determinant on the protein portion of these good acceptors. In contrast the D. discoideum enzyme cannot utilize cathepsin D as a good substrate and, although it phosphorylates uteroferrin efficiently, it does not recognize the protein portion of this acceptor. The oligosaccharide of uteroferrin appears to assume a different conformation than the oligosaccharides of other glycoproteins and glycopeptides, as evidenced by its enhanced sensitivity to mannosidase digestion. This conformation, presumably induced by some interaction with the underlying protein, may be responsible for the specific phosphorylation of uteroferrin by the N-acetylglucosaminylphosphotransferase of D. discoideum.

Acid Phosphatase↗
PubMed1986

Phosphorylation of the oligosaccharide of uteroferrin by UDP-GlcNAc:glycoprotein N-acetylglucosamine-1-phosphotransferases from rat liver, Acanthamoeba castellani, and Dictyostelium discoideum requires alpha 1,2-linked mannose residues.

We have investigated the oligosaccharide requirements of the UDP-GlcNAc:glycoprotein N-acetylglucosamine-1-phosphotransferases from rat liver, Acanthamoeba castellani, and Dictyostelium discoideum. Uteroferrin, an acid hydrolase, was phosphorylated by the three N-acetylglucosaminylphosphotransferases, and the phosphorylated oligosaccharides were isolated and analyzed by ion suppression high performance liquid chromatography. In all three cases, the phosphorylated species contained 6 or more mannose residues. Phosphorylation of the Man5GlcNAc2 oligosaccharide could not be detected even though this was the major species on the native uteroferrin. The Man5GlcNAc2 oligosaccharides lack alpha 1,2-linked mannose residues, whereas the larger oligosaccharides contain 1 or more mannose residues in this linkage. Treatment of intact uteroferrin with an alpha 1,2-specific mannosidase-generated molecules whose oligosaccharides consisted almost entirely of species with 5 mannose residues. The N-acetylglucosaminylphosphotransferases could no longer phosphorylate such molecules. These data indicate that at least 1 alpha 1,2-linked mannose residue must be present on uteroferrin's oligosaccharide for phosphorylation to occur.

Acid Phosphatase↗
PubMed1986

Purification and characterization of UDP-N-acetylgalactosamine: polypeptide N-acetylgalactosaminyltransferase from bovine colostrum and murine lymphoma BW5147 cells.

UDP-GalNAc:polypeptide N-acetylgalactosaminyl-transferase has been purified from two sources. A soluble form, purified 517,000-fold to homogeneity from bovine colostrum, has a molecular mass of 70,000 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 69,000 daltons by gel filtration. A membrane-bound form, partially purified 2,500-fold from BW5147 mouse lymphoma cells, has a molecular mass of 70,000 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 71,500 daltons by gel filtration. The purified colostrum enzyme exhibits specificity for UDP-GalNAc, has its pH optimum between pH 7.2 and 8.6, and requires Mn2+ for activity. The Km is 8 microM for UDP-GalNAc and 2.5 mg/ml for deglycosylated bovine submaxillary mucin. Treatments with endo-beta-N-acetylglucosaminidase H and F indicate that the colostrum enzyme is a glycoprotein containing two N-linked oligosaccharides. On most enzyme molecules, both oligosaccharides are of the complex type, but some molecules contain one complex type and one high mannose type. Antibodies raised against homogeneous bovine enzyme cross-react on immunoblots with a single protein of 71,000 daltons in the partially purified preparation and in a crude microsomal extract from BW5147 cells.

Amino Acids↗
PubMed1986

Deletion of clustered O-linked carbohydrates does not impair function of low density lipoprotein receptor in transfected fibroblasts.

A single exon in the gene for the receptor for plasma low density lipoprotein (LDL) encodes a region of clustered serine and threonine residues that is immediately external to the membrane-spanning sequence. This region has been proposed as the site of clustered O-linked carbohydrate chains. In the current studies we have deleted the 144 base pairs (48 amino acids) that encode this serine- and threonine-rich region from the cDNA for the human LDL receptor. Upon transfection into receptor-deficient hamster fibroblasts, this mutated cDNA encoded a shortened receptor that no longer showed an anomalously high molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Labeling with [3H]glucosamine confirmed the lack of clustered O-linked sugars and further revealed that the shortened receptor and the normal receptor both contained isolated O-linked carbohydrate chains attached to the NH2-terminal portion of the protein. The ratio of clustered to isolated O-linked sugar chains in the normal receptor was estimated to be approximately 4-6 to 1. Despite the loss of clustered O-linked carbohydrate, the LDL receptor encoded by the deletion-bearing cDNA bound and internalized LDL normally. It also recycled normally and exhibited a normal half-life. We conclude that: 1) the serine- and threonine-rich region of the LDL receptor is the site for addition of clustered O-linked carbohydrates; 2) the receptor contains a small number of isolated chains of O-linked carbohydrates in addition to the clustered chains; and 3) the clustered O-linked carbohydrates are not essential for LDL receptor function in cultured hamster fibroblasts.

Amino Acid Sequence↗
PubMed1985

Purification and characterization of a cation-dependent mannose 6-phosphate receptor from murine P388D1 macrophages and bovine liver.

A new binding protein which recognizes the phosphomannosyl recognition marker of lysosomal enzymes has been isolated from P388D1 macrophages which lack the 215-kDa Man-6-P receptor. This receptor was detergent-solubilized from total membranes and purified by chromatography on a lysosomal enzyme affinity column. An identical protein was purified from bovine liver using a two-step procedure which is based on the difference in ion requirement and ligand specificity of the new Man-6-P binding protein and the previously characterized 215-kDa Man-6-P receptor. After detergent solubilization of membranes, both receptors were co-purified on a phosphomannan-Sepharose affinity column run in the presence of MnCl2 and then were separated using a Dictyostelium discoideum lysosomal enzyme-Affi-Gel column which, in the absence of MnCl2, retains only the 215-kDa Man-6-P receptor. The new Man-6-P receptor binds efficiently to phosphomannosyl monoester-containing ligands when MnCl2 is present. This binding is specifically inhibited by Man-6-P or by low pH. The cation-dependent Man-6-P receptor has a subunit molecular size of 46 kDa and appears to be an oligomer composed of three subunits. The receptor contains both high mannose (or hybrid)- and complex-type oligosaccharide units on the basis of sensitivity to digestion with endo-beta-N-acetylglucosaminidase H and endo-beta-N-acetylglucosaminidase F. Radioimmunoassays carried out with two different antibodies demonstrate that the 46-kDa cation-dependent Man-6-P receptor and the 215-kDa cation-independent Man-6-P receptor not only differ in their properties but are also immunologically distinct.

Animals↗
PubMed1985

Non-immune non-activated chicken macrophages destroy murine fibroblasts.

Non-immune non-activated chicken bone marrow-derived macrophages (BM M phi) killed murine embryonic fibroblasts in vitro. Following precultivation for 10-35 days chicken BM M phi had the capacity to destroy normal murine embryonic fibroblasts at effector: target ratios of 10:1 to 1:1. Optimal killing was observed following cocultivation of M phi and fibroblasts for 48-72 hrs. Addition of LPS neither initiated nor potentiated M phi-mediated killing. This study demonstrates that chicken M phi have the capacity to destroy, in vitro, cells of phylogenetically distant species, similarly to the ability of murine M phi to kill chicken fibroblasts. It is suggested that vertebrate M phi xenolytic potential is analogous to the capacity of invertebrate phagocytes to destroy xenografts.

Animals↗
PubMed1985

Lysosomal enzyme binding to mouse P388D1 macrophage membranes lacking the 215-kDa mannose 6-phosphate receptor: evidence for the existence of a second mannose 6-phosphate receptor.

Mouse P388D1 macrophages target newly synthesized acid hydrolases to lysosomes in spite of their lack of the 215-kDa mannose 6-phosphate (Man-6-P) receptor. We now report that these cells contain a membrane-associated Man-6-P receptor that is distinct from the previously described receptor. The new receptor binds lysosomal enzymes containing phosphomannosyl residues. This binding is inhibited by Man-6-P or by pretreatment of the lysosomal enzymes with alkaline phosphatase. Lysosomal enzyme binding occurs at neutral pH and dissociation of the bound ligand occurs at low pH values comparable to those found within endosomes or lysosomes. The new receptor differs from the 215-kDa Man-6-P receptor in two ways. It has an absolute requirement for divalent cations and is unable to bind Dictyostelium discoideum lysosomal enzymes, which contain methylphosphomannosyl residues rather than the usual phosphomannosyl monoesters. Based on the difference in cation requirement, we suggest that the 215-kDa receptor be referred to as Man-6-P receptor CI (cation independent) and the new receptor as Man-6-P receptor CD (cation dependent). We conclude that the Man-6-P receptor CD functions in the targeting of newly synthesized acid hydrolases to lysosomes in P388D1 macrophages.

Animals↗
PubMed1985

Cloning and sequence analysis of cDNA for human cathepsin D.

An 1110-base-pair cDNA clone for human cathepsin D was obtained by screening a lambda gt10 human hepatoma G2 cDNA library with a human renin exon 3 genomic fragment. Poly(A)+ RNA blot analysis with this cathepsin D clone demonstrated a message length of about 2.2 kilobases. The partial clone was used to screen a size-selected human kidney cDNA library, from which two cathepsin D recombinant plasmids with inserts of about 2200 and 2150 base pairs were obtained. The nucleotide sequences of these clones and of the lambda gt10 clone were determined. The amino acid sequence predicted from the cDNA sequence shows that human cathepsin D consists of 412 amino acids with 20 and 44 amino acids in a pre- and a prosegment, respectively. The mature protein region shows 87% amino acid identity with porcine cathepsin D but differs in having nine additional amino acids. Two of these are at the COOH terminus; the other seven are positioned between the previously determined junction for the light and heavy chains of porcine cathepsin D. A high degree of sequence homology was observed between human cathepsin D and other aspartyl proteases, suggesting a conservation of three-dimensional structure in this family of proteins.

Amino Acid Sequence↗
PubMed1985

Lysosomal enzyme phosphorylation in human fibroblasts. Kinetic parameters offer a biochemical rationale for two distinct defects in the uridine diphospho-N-acetylglucosamine:lysosomal enzyme precursor N-acetylglucosamine-1-phosphotransferase.

The primary genetic defect in the lysosomal storage disease mucolipidosis III (ML III) is in the enzyme uridine diphospho-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase. This enzyme has two well-defined functions: specific recognition of lysosomal enzymes (recognition function) and phosphorylation of their oligosaccharides (catalytic function). Using fibroblasts from patients with ML III as the source of enzyme, and alpha-methylmannoside and two lysosomal enzymes as the substrates, we have identified defects in both of these functions. In one group of fibroblasts, the catalytic activity of the N-acetylglucosaminylphosphotransferase is decreased while the ability to recognize lysosomal enzymes as specific substrates remains intact. In the second group of fibroblasts, the ability to recognize lysosomal enzymes is impaired while the catalytic activity of the enzyme is normal. These data provide a biochemical rationale for the previously described genetic heterogeneity among patients with ML III (Honey, N. K., O. T. Mueller, L. E. Little, A. L. Miller, and T. B. Shows, 1982, Proc. Natl. Acad. Sci. USA., 79:7420-7424).

Acid Phosphatase↗
PubMed1984

Lysosomal enzyme phosphorylation. Recognition of a protein-dependent determinant allows specific phosphorylation of oligosaccharides present on lysosomal enzymes.

We have investigated the basis for the specific recognition of lysosomal enzymes by UDP-GlcNAc:lysosomal enzyme N-acetylglucosaminylphosphotransferase. This enzyme is responsible for the selective phosphorylation of mannose residues on lysosomal enzymes. Two mammalian lysosomal enzymes, cathepsin D and uteroferrin, and two nonlysosomal glycoproteins were treated with endo-beta-N-acetylglucosaminidase H to remove those high mannose oligosaccharide units which are accessible on the native protein. These proteins were then tested as inhibitors of three different glycosyltransferases. The endo H-treated lysosomal enzymes were shown to be specific inhibitors of the phosphorylation of intact lysosomal enzymes. Proteolytic fragments of cathepsin D, including the entire light chain and heavy chain, did not retain the ability to be recognized by the N-acetylglucosaminylphosphotransferase. These findings indicate that the intact protein portion of lysosomal enzymes contains a specific recognition determinant which leads to high-affinity binding to the N-acetylglucosaminylphosphotransferase. The expression of this determinant appears to be dependent on the conformation of the protein.

Acetylglucosaminidase↗