Search PubMed⌕ Search

Biomedical subjects

S Kornfeld

Publications and source records attributed to S Kornfeld.

At least 37 records · Page 2Linked to original sources
PubMed1997

Comparative activity of ADP-ribosylation factor family members in the early steps of coated vesicle formation on rat liver Golgi membranes.

We have compared the abilities of mammalian ADP-ribosylation factors (ARFs) 1, 5, and 6 and Saccharomyces cerevisiae ARF2 to serve as substrates for the rat liver Golgi membrane guanine nucleotide exchange factor and to initiate the formation of clathrin- and coatomer protein (COP) I-coated vesicles on these membranes. While Golgi membranes stimulated the exchange of GTPgammaS for GDP on all of the ARFs tested, mammalian ARF1 was the best substrate, with an apparent Km of 5 microM. In all cases myristoylation of ARF was required for stimulation. Agents that inhibit the Golgi membrane guanine nucleotide exchange factor (the fungal metabolite brefeldin A and trypsin treatment) selectively inhibited the guanine nucleotide exchange on mammalian ARF1. Taken together, these data indicate that of the ARFs tested, only mammalian ARF1 is activated efficiently by the Golgi guanine nucleotide exchange factor. The other ARFs are activated mainly by another mechanism, possibly phospholipid-mediated. Once activated, all of the membrane-associated, myristoylated ARFs promoted the recruitment of coatomer to about the same extent. Mammalian ARFs 1 and 5 were the most effective in promoting the recruitment of the AP-1 adaptor complex, whereas yeast ARF2 was the least active. These data indicate that the specificity for ARF action on the Golgi membranes is primarily determined by the Golgi guanine nucleotide exchange factor, which has a strong preference for myristoylated mammalian ARF1.

ADP-Ribosylation Factor 1↗
PubMed1997

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.

Animals↗
PubMed1996

A mannose 6-phosphate-containing N-linked glycopeptide derived from lysosomal acid lipase is bound to MHC class II in B lymphoblastoid cell lines.

Binding of peptides to MHC class II Ag generates ligands for TCR of Th lymphocytes. We have identified a novel class of peptides bound to MHC class II: mannose 6- phosphate (Man-6-P) containing glycopeptides from lysosomal enzymes. These species were identified in the process of characterizing mannose 6-phosphate/insulin-like growth factor II (M-6-P/IGF-II) receptor binding to the surface of B lymphoblasts. Surface iodination and Man-6-P/IGF-II receptor affinity chromatography implicated MHC class II as a carrier of Man-6-P-modified oligosaccharides. These oligosaccharides were found to be primarily associated with the bound peptide. Peptides eluted from the Man-6-P/IGF-II receptor-binding fraction of immunoaffinity-purified MHC class II from the Swei cell line contained a sequence derived from the propiece of lysosomal acid lipase. Partial sequences were also obtained for peptides from other HLA-DR alleles but none of these were attributable to known proteins. This study defines a novel approach for isolating rare glycan-modified peptides from MHC class II and demonstrates that very large secondary modifications are tolerated in peptides bound to MHC class II.

Amino Acid Sequence↗
PubMed1996

The targeting of Lamp1 to lysosomes is dependent on the spacing of its cytoplasmic tail tyrosine sorting motif relative to the membrane.

Lamp1 is a type I transmembrane glycoprotein that is localized primarily in lysosomes and late endosomes. Newly synthesized molecules are mostly transported from the trans-Golgi network directly to endosomes and then to lysosomes. A minor pathway involves transport via the plasma membrane. The 11-amino acid cytoplasmic tail of lamp1 contains a tyrosine-based motif that has been previously shown to mediate sorting in the trans-Golgi network and rapid internalization at the plasma membrane. We studied whether this motif also mediates sorting in endosomes. We found that mutant forms of lamp1 in which all the amino acids of the cytoplasmic tail were modified except for the RKR membrane anchor and the YXXI sorting motif still localized to dense lysosomes, indicating that the YXXI motif is sufficient to confer proper intracellular targeting. However, when the spacing of the YXXI motif relative to the membrane was changed by deleting one amino acid or adding five amino acids, lysosomal targeting was almost completely abolished. Kinetic studies showed that these mutants were trapped in a recycling pathway, involving trafficking between the plasma membrane and early endocytic compartments. These findings indicate that the YXXI signal of lamp1 is recognized at several sorting sites, including the trans-Golgi network, the plasma membrane, and the early/sorting endosomes. Small changes in the spacing of this motif relative to the membrane dramatically impair sorting in the early/sorting endosomes but have only a modest effect on internalization at the plasma membrane. The spacing of sorting signals relative to the membrane may prove to be an important determinant in the functioning of these signals.

Amino Acid Sequence↗
PubMed1996

Cysteine34 of the cytoplasmic tail of the cation-dependent mannose 6-phosphate receptor is reversibly palmitoylated and required for normal trafficking and lysosomal enzyme sorting.

We have examined whether the two cysteine residues (Cys30 and Cys34) in the cytoplasmic tail of the cation-dependent mannose 6-phosphate receptor are palmitoylated via thioesters and whether these residues influence the biologic function of the receptor. To do this, mouse L cells expressing wild-type and mutant receptors were analyzed by metabolic labeling with [3H]palmitate, immunoprecipitation, and SDS-PAGE. Both Cys30 and Cys34 were found to be sites of palmitoylation and together they accounted for the total palmitoylation of the receptor. The palmitate rapidly turned over with a half-life of approximately 2 h compared to a half-life of greater than 40 h for the protein. Mutation of Cys34 to Ala resulted in the gradual accumulation of the receptor in dense lysosomes and the total loss of cathepsin D sorting function in the Golgi. A Cys30 to Ala mutation had no biologic consequences, showing the importance of Cys34. Mutation of amino acids 35-39 to alanines impaired palmitoylation of Cys30 and Cys34 and resulted in abnormal receptor trafficking to lysosomes and loss of cathepsin D sorting. These data suggest that palmitoylation of Cys30 and Cys34 leads to anchoring of this region of the cytoplasmic tail to the lipid bilayer. Anchoring via Cys34 is essential for the normal trafficking and lysosomal enzyme sorting function of the receptor.

Amino Acid Sequence↗
PubMed1996

The biogenesis of the MHC class II compartment in human I-cell disease B lymphoblasts.

The localization and intracellular transport of major histocompatibility complex (MHC) class II molecules nd lysosomal hydrolases were studied in I-Cell Disease (ICD) B lymphoblasts, which possess a mannose 6-phosphate (Man-6-P)-independent targeting pathway for lysosomal enzymes. In the trans-Golgi network (TGN), MHC class II-invariant chain complexes colocalized with the lysosomal hydrolase cathepsin D in buds and vesicles that lacked markers of clathrin-coated vesicle-mediated transport. These vesicles fused with the endocytic pathway leading to the formation of "early" MHC class II-rich compartments (MIICs). Similar structures were observed in the TGN of normal beta lymphoblasts although they were less abundant. Metabolic labeling and subcellular fractionation experiments indicated that newly synthesized cathepsin D and MHC class II-invariant chain complexes enter a non-clathrin-coated vesicular structure after their passage through the TGN and segregation from the secretory pathway. These vesicles were also devoid of the cation-dependent mannose 6-phosphate (Man-6-P) receptor, a marker of early and late endosomes. These findings suggest that in ICD B lymphoblasts the majority of MHC class II molecules are transported directly from the TGN to "early" MIICs and that acid hydrolases cam be incorporated into MIICs simultaneously by a Man-6-P-independant process.

Antigens, Differentiation, B-Lymphocyte↗
PubMed1996

AP-2-containing clathrin coats assemble on mature lysosomes.

Coat proteins appear to play a general role in intracellular protein trafficking by coordinating a membrane budding event with cargo selection. Here we show that the AP-2 adaptor, a clathrin-associated coat-protein complex that nucleates clathrin-coated vesicle formation at the cell surface, can also initiate the assembly of normal polyhedral clathrin coats on dense lysosomes under physiological conditions in vitro. Clathrin coat formation on lysosomes is temperature dependent, displays an absolute requirement for ATP, and occurs in both semi-intact cells and on purified lysosomes, suggesting that clathrin-coated vesicles might regulate retrograde membrane traffic out of the lysosomal compartment.

Animals↗
PubMed1996

Cystic fibrosis heterozygote screening in 5,161 pregnant women.

A screening program for cystic fibrosis (CF) heterozygotes was conducted in a large HMO prenatal population, to evaluate the level of interest among eligible patients, the effectiveness of prescreening education, attitudes toward the screening process, psychological effects, and utilization of prenatal diagnosis and its outcomes. The heterozygote identification rate and frequency of specific CFTR mutations were also assessed. Identified carriers were offered genetic counseling and testing of male partners. Prenatal diagnosis was offered if both parents were identified as carriers. A total of 5,161 women underwent carrier testing; 947 others completed survey instruments only. The acceptance rate of screening was high (78%), and pretest education by videotape was generally effective. Adverse psychological effects were not reported. Participants generally found screening to be desirable and useful. Screening identified 142 female heterozygotes, 109 couples in which the male partner was not a carrier, and 7 high-risk couples. The incidence of R117H mutations was much higher than expected. The number of identified carriers was much lower in Hispanics than in Caucasians. We conclude that large-scale prenatal screening for CF heterozygotes in the absence of a family history of CF is an acceptable method for identifying couples at risk for affected fetuses. Sufficient pretest education can be accomplished efficiently, test insensitivity is well accepted, adverse psychological events are not observed, and general patient satisfaction is high.

Adult↗
PubMed1995

Determination of the structural requirements for palmitoylation of p63.

Palmitoylation of p63, a type II membrane protein localized in the endoplasmic reticulum, is induced in a reversible manner by the drug brefeldin A. To study the requirements for palmitoylation, mutant forms of p63 were expressed in COS cells and analyzed by metabolic labeling with [3H]palmitate, immunoprecipitation, and SDS-polyacrylamide gel electrophoresis. By investigating deletion and point mutations, Cys100 in the 106-amino acid cytoplasmic tail of p63 has been identified as the site of acylation. Site-directed mutagenesis of residues 99-105 together with cytoplasmic tail truncation mutants showed that the amino acids surrounding Cys100 are not critical for palmitoylation of this residue. Analysis of a chimeric construct between p63 and the plasma membrane protein dipeptidylpeptidase IV further revealed that p63 palmitoylation is not dependent on its transmembrane domain. In contrast, the six-amino acid distance between the end of the predicted transmembrane domain and the palmitoylation site was found to be essential for proper acylation of p63.

Amino Acid Sequence↗
PubMed1995

Different domains of the AP-1 adaptor complex are required for Golgi membrane binding and clathrin recruitment.

The assembly of clathrin-coated buds on the Golgi requires the recruitment of the heterotetrameric AP-1 adaptor complex, which is dependent on both guanine nucleotides and the small GTP-binding protein ADP-ribosylation factor (ARF). Here, we have investigated the structural domains of the AP-1 complex necessary for ARF-mediated translocation of the adaptor complex onto Golgi membranes and the subsequent recruitment of clathrin onto the membrane. Controlled proteolysis of purified AP-1, derived from bovine adrenal coated vesicles, was used to generate AP-1 core fragments composed of the amino-terminal trunk regions of the beta 1 and gamma subunits and associated mu 1 and sigma 1 subunits, and lacking either the beta 1 subunit carboxyl-terminal appendage or both beta 1 and gamma subunit appendages. On addition of these truncated fragments to AP-1-depleted adrenal cytosol, both types of core fragments were efficiently recruited onto Golgi membranes in the presence of GTP gamma S. Recruitment of both core fragments was inhibited by the fungal metabolite brefeldin A, indicative of an ARF-dependent process. Limited tryptic digestion of recruited, intact cytosolic AP-1 resulted in the quantitative release of the globular carboxyl-terminal appendage domains of the beta 1 and gamma subunits. The adaptor core complex remained associated with the Golgi membranes. Recruitment of cytosolic clathrin onto the Golgi membranes was strictly dependent on the presence of intact AP-1. Tryptic removal of the beta 1 subunit appendage prevented subsequent clathrin recruitment. We conclude that the structural determinants required for the ARF-mediated binding of cytosolic AP-1 onto Golgi membranes are contained within the adaptor core, and that the carboxyl-terminal appendage domains of the beta 1 and gamma subunits do not play any role in this process. Subsequent recruitment of cytosolic clathrin, however, requires an intact beta 1 subunit.

3T3 Cells↗
PubMed1995

A novel mutagenesis strategy identifies distantly spaced amino acid sequences that are required for the phosphorylation of both the oligosaccharides of procathepsin D by N-acetylglucosamine 1-phosphotransferase.

A novel combinatorial mutagenesis strategy (shuffle mutagenesis) was developed to identify sequences in the propiece and amino lobe of cathepsin D which direct oligosaccharide phosphorylation by UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine 1-phosphotransferase. Propiece restriction fragments and oligonucleotide cassettes corresponding to 13 regions of the cathepsin D and glycopepsinogen amino lobes were randomly shuffled together to generate a large library of chimeric molecules. The library was inserted into an expression vector encoding the carboxyl lobe of cathepsin D with a carboxyl-terminal myc epitope and a CD8 transmembrane extension. Transfected COS1 cells expressing the membrane-anchored forms of the cathepsin D/glycopepsinogen chimeras at the cell surface were selected with solid phase mannose 6-phosphate receptor or an antibody to the myc epitope. Plasmids were rescued in Escherichia coli and sequenced by hybridization to the original oligonucleotide cassettes. Two regions of the cathepsin D amino lobe (segments 7 and 12) were found to contribute to proper folding, surface expression, and selective phosphorylation of the carboxyl lobe oligosaccharide. Two different cathepsin D regions (the propiece and segment 5) cooperated with a previously identified recognition element in the carboxyl lobe to allow efficient phosphorylation of both the amino and carboxyl lobe oligosaccharides. Three general models for extending the catalytic reach of N-acetylglucosamine 1-phosphotransferase to widely spaced oligosaccharides are presented.

Amino Acid Sequence↗
PubMed1995

A determinant in the cytoplasmic tail of the cation-dependent mannose 6-phosphate receptor prevents trafficking to lysosomes.

The bovine cation-dependent mannose 6-phosphate receptor (CD-MPR) is a type 1 transmembrane protein that cycles between the trans-Golgi network, endosomes, and the plasma membrane. When the terminal 40 residues were deleted from the 67-amino acid cytoplasmic tail of the CD-MPR, the half-life of the receptor was drastically decreased and the mutant receptor was recovered in lysosomes. Analysis of additional cytoplasmic tail truncation mutants and alanine-scanning mutants implicated amino acids 34-39 as being critical for avoidance of lysosomal degradation. The cytoplasmic tail of the CD-MPR was partially effective in preventing the lysosomal membrane protein Lamp1 from entering lysosomes. Complete exclusion required both the CD-MPR cytoplasmic tail and transmembrane domain. The transmembrane domain alone had just a minor effect on the distribution of Lamp1. These findings indicate that the cytoplasmic tail of the CD-MPR contains a signal that prevents the receptor from trafficking to lysosomes. The transmembrane domain of the CD-MPR also contributes to this function.

Amino Acid Sequence↗
PubMed1995

Reassessment of the subcellular localization of p63.

p63 is a type II integral membrane protein that has previously been suggested to be a resident protein of a membrane network interposed between the ER and the Golgi apparatus. In the present study, we have produced a polyclonal antibody against the purified human p63 protein to reassess the subcellular distribution of p63 by confocal immunofluorescence, immunoelectron microscopy, and cell fractionation. Double immunofluorescence of COS cells showed significant colocalization of p63 and a KDEL-containing lumenal ER marker protein, except for differences in the staining of the outer nuclear membrane. Immunoelectron microscopy of native HepG2 cells and of COS cells transfected with p63 revealed that both endogenous and overexpressed p63 are predominantly localized in the rough ER. While p63 was colocalized with protein disulfide isomerase, an ER marker protein, very little overlap of p63 was found with ERGIC-53, an established marker for the ER-Golgi intermediate compartment. When rough and smooth membranes were prepared from rat liver, p63 was found to copurify with ribophorin II, a rough ER protein. Both p63 and ribophorin II were predominantly recovered in rough microsomes and were largely separated from the intermediate compartment marker protein p58. From these results it is concluded that p63 is localized in the rough ER.

Amino Acid Sequence↗
PubMed1995

Phase Ia trial of murine immunoglobulin A antitransferrin receptor antibody 42/6.

In preclinical in vitro and in vivo systems, mAbs to human transferrin (Tf) receptors blocked iron uptake from Tf and showed antitumor activity. However, Tf receptors are also displayed by normal tissues, and a large, soluble pool of circulating serum Tf receptors has been detected. We report results of a Phase Ia trial of IgA monoclonal anti-Tf receptor antibody 42/6. Twenty-seven patients with advanced refractory cancer received 33 treatments with 42/6 administered as a 24-h infusion at doses ranging from 2.5 to 300 mg/m2. 42/6 was generally well tolerated, although one patient receiving a second treatment experienced an allergic-type response associated with a human antimouse antibody response. Three patients with hematological cancers showed mixed tumor responses; there were no partial or complete remissions. Peak serum levels of antibody were obtained at the termination of the 24-h infusion. At doses >/=25 mg/m2, there was a linear relationship between the 42/6 dose and average peak serum 42/6 levels ranging from <1 to 36 microgram/ml. Serum Tf receptors showed a dose-dependent decrease during 42/6 infusion to 20-30% of baseline, and remained depressed for at least 48 h after terminating the infusion. Serum 42/6 levels rose in an inverse relationship to the drop in Tf receptors. 42/6 induced an increase in serum iron and Tf saturation consistent with blockade of peripheral iron uptake, and reduced Tf receptor display by bone marrow cells. Human antimouse antibody was detected in nine patients. Anti-Tf receptor antibody was well tolerated and mediated in vivo effects on iron uptake and Tf display. Antibody concentrations capable of inhibiting malignant blood cell growth were obtained without toxicity. This represents the first clinical trial of an IgA mouse mAb, and one of only a few trials in which an antibody reacting with a broad range of normal tissues has been administered. Additional clinical trials of anti-Tf receptor antibodies in blood cell cancers are indicated.

Adult↗
PubMed1994

Subcellular localization and targeting of cathepsin E.

The subcellular distribution and targeting of the non-lysosomal aspartic proteinase cathepsin E have been studied using mouse L cells and monkey Cos 1 cells that were transfected with cDNA encoding cathepsin E. The cathepsin E was retained in L cells for at least 20 h without significant degradation and its single N-linked oligosaccharide remained sensitive to endo-beta-N-acetyl-glucosaminidase H. When cathepsin E was overexpressed by transient transfection in Cos 1 cells, it was very slowly secreted into the media. The intracellular form of the enzyme contained a high mannose oligosaccharide which was processed to a complex type species upon secretion. In double label immunofluorescence studies, cathepsin E co-localized with cathepsin D-myc-KDEL, an endoplasmic reticulum (ER) marker. Subcellular fractionation on a Percoll density gradient showed that the cathepsin E co-migrated with membranous vesicles that were distinct from dense lysosomes. Only a trace amount of the enzyme was recovered in the soluble fraction. These findings indicate that in L cells and Cos 1 cells, the intracellular location of cathepsin E is the endoplasmic reticulum. To identify the protein sequences required for ER retention, we made chimeric proteins between cathepsin E and pepsinogen, an aspartic proteinase that is rapidly secreted by Cos 1 cells. We found that amino acids 1-48 of cathepsin E are important for its retention in the ER. Within this region, Cys7, which is involved in covalent dimer formation, plays a significant role in the retention.

Alternative Splicing↗
PubMed1994

Herpes simplex virus glycoprotein D acquires mannose 6-phosphate residues and binds to mannose 6-phosphate receptors.

Herpes simplex viruses (HSV) use multiple and sequential receptors to enter host cells. HSV glycoprotein D (gD) has been implicated in binding to cellular receptors that facilitate virus penetration into cells. We used soluble forms of gD that were expressed in Chinese hamster ovary cells to characterize and identify a putative cellular receptor for HSV as the 275-kDa mannose 6-phosphate/insulin-like growth factor II receptor. Soluble gD also bound to the 46-kDa cation-dependent mannose 6-phosphate (Man-6-P) receptor and was extensively modified with Man-6-P residues on its Asn-linked oligosaccharides. Additionally, soluble gD was a high affinity substrate for N-acetylglucosamine-1-phosphotransferase, the first enzyme in the biosynthetic pathway for the addition of Man-6-P residues to lysosomal enzymes. The membrane form of gD immunoprecipitated from HSV-infected cells also bound to the 275-kDa mannose 6-phosphate/insulin-like growth factor II receptor, albeit poorly, and only a small fraction of the membrane gD was modified with Man-6-P. Notwithstanding this low level of mannose phosphorylation, the interaction between gD and Man-6-P receptors may play a role in some aspect of virus entry or egress.

Amino Acid Sequence↗