Biomedical subjects

Stuart Kornfeld

Publications and source records attributed to Stuart Kornfeld.

18 recordsLinked to original sources

PubMed2006

Selective action of the iminosugar isofagomine, a pharmacological chaperone for mutant forms of acid-beta-glucosidase.

Gaucher disease is a lysosomal glycolipid storage disorder characterized by defects in acid-beta-glucosidase (GlcCerase), the enzyme responsible for the catabolism of glucosylceramide. We recently demonstrated that isofagomine (IFG), an iminosugar that binds to the active site of GlcCerase, enhances the folding, transport and activity of the N370S mutant form of GlcCerase. In this study we compared the effects of IFG on a number of other glucosidases and glucosyltransferases. We report that IFG has little or no inhibitory activity towards intestinal disaccharidase enzymes, ER alpha-glucosidase II or glucosylceramide synthase at concentrations previously shown to enhance N370S GlcCerase folding and trafficking in Gaucher fibroblasts. Furthermore, treatment of wild type fibroblasts with high doses of IFG did not alter the processing of newly synthesized N-linked oligosaccharides. These findings support further evaluation of IFG as a potential therapeutic agent in the treatment of some forms of Gaucher disease.

1-Deoxynojirimycin↗

PubMed2006

COG-7-deficient Human Fibroblasts Exhibit Altered Recycling of Golgi Proteins.

Recently, we reported that two siblings presenting with the clinical syndrome congenital disorders of glycosylation (CDG) have mutations in the gene encoding Cog7p, a member of the conserved oligomeric Golgi (COG) complex. In this study, we analyzed the localization and trafficking of multiple Golgi proteins in patient fibroblasts under a variety of conditions. Although the immunofluorescent staining pattern of several Golgi proteins was indistinguishable from normal, the staining of endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC)-53 and the vesicular-soluble N-ethylmaleimide-sensitive factor attachment protein receptors GS15 and GS28 was abnormal, and the steady-state level of GS15 was greatly decreased. Retrograde transport of multiple Golgi proteins to the ER in patient fibroblasts via brefeldin A-induced tubules was significantly slower than occurs in normal fibroblasts, whereas anterograde protein trafficking was much less affected. After prolonged treatment with brefeldin A, several Golgi proteins were detected in clusters that colocalize with the microtubule-organizing center in patient cells. All of these abnormalities were normalized in COG7-corrected patient fibroblasts. These results serve to better define the role of the COG complex in facilitating protein trafficking between the Golgi and ER and provide a diagnostic framework for the identification of CDG defects involving trafficking proteins.

Adaptor Proteins, Vesicular Transport↗

PubMed2005

Identification of the minimal lysosomal enzyme recognition domain in cathepsin D.

Specific recognition of lysosomal hydrolases by UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase, the initial enzyme in the biosynthesis of mannose 6-phosphate residues, is governed by a common protein determinant. Previously, we generated a lysosomal enzyme recognition domain in the secretory protein glycopepsinogen by substituting in two regions (lysine 203 and amino acids 265-293 of the beta loop) from cathepsin D, a highly related lysosomal protease. Here we show that substitution of just two lysines (Lys-203 and Lys-267) stimulates mannose phosphorylation 116-fold. Substitution of additional residues in the beta loop, particularly lysines, increased phosphorylation 4-fold further, approaching the level obtained with intact cathepsin D. All the phosphorylation occurred at the carboxyl lobe glycan, indicating that additional elements are required for phosphorylation of the amino lobe glycan. These data support the proposal that as few as two lysines in the correct orientation to each other and to the glycan can serve as the minimal elements of the lysosomal enzyme recognition domain. However, our findings show that the spacing between lysines is flexible and other residues contribute to the recognition marker.

Amino Acid Sequence↗

PubMed2005

A splicing mutation in the alpha/beta GlcNAc-1-phosphotransferase gene results in an adult onset form of mucolipidosis III associated with sensory neuropathy and cardiomyopathy.

A 47-year-old female who presented with a dilated cardiomyopathy and mild neuropathy was found to have pseudoHurler polydystrophy (mucolipidosis III). The serum lysosomal enzymes were strikingly elevated and GlcNAc-1-phosphotransferase activity in the patient's fibroblasts was 3% of normal. Sequence analysis of the patient's genomic DNA revealed a homozygous mutation of the last nucleotide of the 135-bp exon 7 of the phosphotransferase gene encoding the alpha/beta subunits, resulting in aberrant splicing and skipping of this exon. Remarkably, none of the skeletal and connective tissue anomalies characteristic of the disease were present. This case is the first example of mucolipidosis III presenting in an adult patient and further broadens the clinical spectrum of the disease.

Age of Onset↗

PubMed2004

The cytoplasmic tail of the cation-independent mannose 6-phosphate receptor contains four binding sites for AP-1.

The trafficking of the cation-independent mannose 6-phosphate receptor between the trans-Golgi network and endosomes requires binding of sorting determinants in the cytoplasmic tail of the receptor to adaptor protein complex-1 (AP-1). Using a GST pull-down binding assay, four binding motifs were identified in the cytoplasmic tail: a tyrosine-based motif ((26)YSKV(29)), an internal dileucine-based motif ((39)ETEWLM(44)), and two casein kinase 2 sites ((84)DSEDE(88) and (154)DDSDED(159)). The YSKV motif mediated the strongest interaction with AP-1 and the two CK2 motifs bound AP-1 only when they were phosphorylated. The COOH-terminal dileucines were not required for interaction with AP-1.

Adaptor Protein Complex 1↗

PubMed2004

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.

Base Sequence↗

PubMed2004

GGA1 interacts with the adaptor protein AP-1 through a WNSF sequence in its hinge region.

The Golgi-associated gamma-adaptin-related ADP-ribosylation factor-binding proteins (GGAs) are critical components of the transport machinery that mediates the trafficking of the mannose 6-phosphate receptors and associated cargo from the trans-Golgi network to the endosomes. The GGAs colocalize in vivo with the clathrin adaptor protein AP-1 and bind to AP-1 in vitro, suggesting that the two proteins may cooperate in packaging the mannose 6-phosphate receptors into clathrin-coated vesicles at the trans-Golgi network. Here, we demonstrate that the sequence, (382)WNSF(385), in the hinge region of GGA1 mediates its interaction with the AP-1 gamma-ear. The Trp and Phe constitute critical amino acids in this interaction. The binding of Rabaptin5 to the AP-1 gamma-ear, which occurs through a FXXPhi motif, is inhibited by a peptide encoding the GGA1 (382)WNSF(385) sequence. Moreover, mutations in the AP-1 gamma-ear that abolish its interaction with Rabaptin5 also preclude its association with GGA1. These results suggest that the GGA1 WXXF-type and Rabaptin5 FXXPhi-type motifs bind to the same or highly overlapping sites in the AP-1 gamma-ear. This binding is modulated by residues adjacent to the core motifs.

ADP-Ribosylation Factors↗

PubMed2004

The GGA proteins: key players in protein sorting at the trans-Golgi network.

The GGA (Golgi-localized, gamma-ear containing, ADP-ribosylation factor binding) family of multidomain coat proteins was first described in the year 2000. They are now known to occupy a central position in the trafficking of the mannose 6-phosphate receptors and other cargo molecules from the trans-Golgi network to the endosome/lysosome system. This review covers the recent structural and cell biological studies that have provided mechanistic insights into the function of the GGAs in mannose 6-phosphate receptor trafficking.

ADP-Ribosylation Factors↗

PubMed2004

Blasts from the past.

With this issue of the JCI, we celebrate the 80th anniversary of the Journal. While 80 years is not a century, we still feel it is important to honor what the JCI has meant to the biomedical research community for 8 decades. To illustrate why the JCI is the leading general-interest translational research journal edited by and for biomedical researchers, we have asked former JCI editors-in-chief to reflect on some of the major scientific advances reported in the pages of the Journal during their tenures.

Animals↗

PubMed2003

Mammalian GGAs act together to sort mannose 6-phosphate receptors.

The GGAs (Golgi-localized, gamma ear-containing, ADP ribosylation factor-binding proteins) are multidomain proteins implicated in protein trafficking between the Golgi and endosomes. We examined whether the three mammalian GGAs act independently or together to mediate their functions. Using cryo-immunogold electron microscopy, the three GGAs were shown to colocalize within coated buds and vesicles at the trans-Golgi network (TGN) of HeLa cells. In vitro binding experiments revealed multidomain interactions between the GGAs, and chemical cross-linking experiments demonstrated that GGAs 1 and 2 form a complex on Golgi membranes. RNA interference of each GGA resulted in decreased levels of the other GGAs and their redistribution from the TGN to cytosol. This was associated with impaired incorporation of the cation-independent mannose 6-phosphate receptor into clathrin-coated vesicles at the TGN, partial redistribution of the receptor to endosomes, and missorting of cathepsin D. The morphology of the TGN was also altered. These findings indicate that the three mammalian GGAs cooperate to sort cargo and are required for maintenance of TGN structure.

ADP-Ribosylation Factors↗

PubMed2003

Endoplasmic reticulum-associated degradation of mammalian glycoproteins involves sugar chain trimming to Man6-5GlcNAc2.

Endoplasmic reticulum-associated degradation of misfolded or misprocessed glycoproteins in mammalian cells is prevented by inhibitors of class I alpha-mannosidases implicating mannose trimming from the precursor oligosaccharide Glc3Man9GlcNAc2 as an essential step in this pathway. However, the extent of mannose removal has not been determined. We show here that glycoproteins subject to endoplasmic reticulum-associated degradation undergo reglucosylation, deglucosylation, and mannose trimming to yield Man6GlcNAc2 and Man5GlcNAc2. These structures lack the mannose residue that is the acceptor of glucose transferred by UDP-Glc:glycoprotein glucosyltransferase. This could serve as a mechanism for removal of the glycoproteins from folding attempts catalyzed by cycles of reglucosylation and calnexin/calreticulin binding and result in targeting of these molecules for proteasomal degradation.

3T3 Cells↗

PubMed2003

AP-1 binding to sorting signals and release from clathrin-coated vesicles is regulated by phosphorylation.

The adaptor protein complex-1 (AP-1) sorts and packages membrane proteins into clathrin-coated vesicles (CCVs) at the TGN and endosomes. Here we show that this process is highly regulated by phosphorylation of AP-1 subunits. Cell fractionation studies revealed that membrane-associated AP-1 differs from cytosolic AP-1 in the phosphorylation status of its beta1 and mu1 subunits. AP-1 recruitment onto the membrane is associated with protein phosphatase 2A (PP2A)-mediated dephosphorylation of its beta1 subunit, which enables clathrin assembly. This Golgi-associated isoform of PP2A exhibits specificity for phosphorylated beta1 compared with phosphorylated mu1. Once on the membrane, the mu1 subunit undergoes phosphorylation, which results in a conformation change, as revealed by increased sensitivity to trypsin. This conformational change is associated with increased binding to sorting signals on the cytoplasmic tails of cargo molecules. Dephosphorylation of mu1 (and mu2) by another PP2A-like phosphatase reversed the effect and resulted in adaptor release from CCVs. Immunodepletion and okadaic acid inhibition studies demonstrate that PP2A is the cytosolic cofactor for Hsc-70-mediated adaptor uncoating. A model is proposed where cyclical phosphorylation/dephosphorylation of the subunits of AP-1 regulate its function from membrane recruitment until its release into cytosol.

Adaptor Protein Complex 1↗

PubMed2003

Phosphorylation-induced conformational changes regulate GGAs 1 and 3 function at the trans-Golgi network.

The GGAs (Golgi-localizing, gamma-adaptin ear homology domain, ARF-binding) are a family of multidomain proteins implicated in protein trafficking between the Golgi and the endosomes. All three GGAs (1, 2, and 3) bind to the mannose 6-phosphate receptor tail via their VHS domains, as well as to the adaptor protein complex-1 via their hinge domains. The latter interaction has been proposed to be important for cooperative packaging of cargo into forming clathrin-coated carriers at the trans-Golgi network. Here we present evidence that GGA1 function is highly regulated by cycles of phosphorylation and dephosphorylation. Cell fractionation showed that the phosphorylated pool of GGA1 resided predominantly in the cytosol and that recruitment onto membranes was associated with dephosphorylation. Okadaic acid inhibition studies and in vitro dephosphorylation assays indicated that dephosphorylation is mediated by a protein phosphatase 2A-like phosphatase. Dephosphorylation of GGA1 induced a change in the conformation to an "open" form as measured by gel filtration and sucrose gradient analyses. This was associated with enhanced binding to ligands because of release of autoinhibition and increased binding to the adaptor protein complex-1 gamma-appendage. A model is proposed for the regulation of GGA1 function at the trans-Golgi network.

ADP-Ribosylation Factors↗

PubMed2003

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.

Amino Acid Motifs↗

PubMed2002

Cooperation of GGAs and AP-1 in packaging MPRs at the trans-Golgi network.

The Golgi-localized, gamma-ear-containing, adenosine diphosphate ribosylation factor-binding proteins (GGAs) are multidomain proteins that bind mannose 6-phosphate receptors (MPRs) in the Golgi and have an essential role in lysosomal enzyme sorting. Here the GGAs and the coat protein adaptor protein-1 (AP-1) were shown to colocalize in clathrin-coated buds of the trans-Golgi networks of mouse L cells and human HeLa cells. Binding studies revealed a direct interaction between the hinge domains of the GGAs and the gamma-ear domain of AP-1. Further, AP-1 contained bound casein kinase-2 that phosphorylated GGA1 and GGA3, thereby causing autoinhibition. This could induce the directed transfer of the MPRs from GGAs to AP-1. MPRs that are defective in binding to GGAs are poorly incorporated into AP-1-containing clathrin-coated vesicles. Thus, the GGAs and AP-1 interact to package MPRs into AP-1-containing coated vesicles.

ADP-Ribosylation Factors↗

PubMed2002

Human mannose 6-phosphate-uncovering enzyme is synthesized as a proenzyme that is activated by the endoprotease furin.

N-Acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase, also known as "uncovering" enzyme (UCE), is localized in the trans-Golgi network, where it removes a covering N-acetylglucosamine from the mannose 6-phosphate recognition marker on lysosomal acid hydrolases. Here we show that UCE is synthesized as an inactive proenzyme that is activated by the endoprotease furin, which cleaves an RARLPR/D sequence to release a 24-amino acid propiece. As furin is localized in the trans-Golgi network, newly synthesized UCE is inactive until it reaches this terminal Golgi compartment. LoVo cells (derived from a human colon adenocarcinoma) lack furin activity and have extremely low UCE activity. Addition of furin to LoVo cell extracts restores UCE activity to normal levels, demonstrating that the UCE proenzyme is stable in this cell type. LoVo cells secrete acid hydrolases with phosphomannose diesters as a consequence of the deficient UCE activity. This demonstrates for the first time that UCE is the only enzyme in these cells capable of efficiently uncovering phosphomannose diesters. UCE also hydrolyzes UDP-GlcNAc, a sugar donor for Golgi N-acetylglucosaminyltransferases. The fact that UCE is not activated until it reaches the trans-Golgi network may ensure that the pool of UDP-GlcNAc in the Golgi stack is not depleted, thereby maintaining proper oligosaccharide assembly.

Amino Acid Sequence↗

PubMed2002

Interaction of the cation-dependent mannose 6-phosphate receptor with GGA proteins.

The GGAs (Golgi-localizing, gamma-adaptin ear homology domain, ARF-binding) are a multidomain family of proteins implicated in protein trafficking between the Golgi and endosomes. Recent evidence has established that the cation-independent (CI) and cation-dependent (CD) mannose 6-phosphate receptors (MPRs) bind specifically to the VHS domains of the GGAs through acidic cluster-dileucine motifs at the carboxyl ends of their cytoplasmic tails. However, the CD-MPR binds the VHS domains more weakly than the CI-MPR. Alignment of the C-terminal residues of the two receptors revealed a number of non-conservative differences in the acidic cluster-dileucine motifs and the flanking residues. Mutation of these residues in the CD-MPR cytoplasmic tail to the corresponding residues in the CI-MPR conferred either full binding (H63D mutant), intermediate binding (R60S), or unchanged binding (E56F/S57H) to the GGAs as determined by in vitro glutathione S-transferase pull-down assays. Furthermore, the C-terminal methionine of the CD-MPR, but not the C-terminal valine of the CI-MPR, inhibited GGA binding. Addition of four alanines to the C-terminal valine of the CI-MPR also severely reduced GGA binding, demonstrating the importance of the spacing of the acidic cluster-dileucine motif relative to the C terminus for optimal GGA interaction. Mouse L cells stably expressing CD-MPRs with mutations that enhance GGA binding sorted cathepsin D more efficiently than wild-type CD-MPR. These studies provide an explanation for the observed differences in the relative affinities of the two MPRs for the GGA proteins. Furthermore, they indicate that the GGAs participate in lysosomal enzyme sorting mediated by the CD-MPR.

ADP-Ribosylation Factors↗

PubMed2001

Multiple signals regulate trafficking of the mannose 6-phosphate-uncovering enzyme.

The "uncovering enzyme," which catalyzes the second step in the formation of the mannose 6-phosphate recognition marker on lysosomal enzyme oligosaccharides, resides primarily in the trans-Golgi network and cycles between this compartment and the plasma membrane. An analysis of green fluorescent protein-uncovering enzyme chimeras revealed that the transmembrane segment and the first 11 residues of the 41-residue-cytoplasmic tail are sufficient for retention in the trans-Golgi network. The next eight residues ((486)YAYHPLQE(493)) facilitate exit from this compartment. Kinetic studies demonstrated that the (488)YHPL(491) sequence also mediates rapid internalization at the plasma membrane. This motif binds adaptor protein-2 in glutathione S-transferase-uncovering enzyme-cytoplasmic tail pull-down assays, indicating that the uncovering enzyme is endocytosed via clathrin-coated vesicles. Consistent with this finding, endogenous uncovering enzyme was detected in purified clathrin-coated vesicles. The enzyme with a Y486A mutation is internalized normally but accumulates on the cell surface because of increased recycling to the plasma membrane. This residue is required for efficient return of the enzyme from endosomes to the trans-Golgi network. These findings indicate that the YAYHPLQE motif is recognized at several sorting sites, including the trans-Golgi network, the plasma membrane, and the endosome.

Adrenal Glands↗