S Kornfeld

Washington University in St. Louis, San Luis, Missouri, United States

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Publications (232)1524.09 Total impact

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    Full-text · Article · Dec 2015 · Glycobiology
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    ABSTRACT: The lysosomal enzyme receptor protein (LERP) of Drosophila melanogaster is the ortholog of the mammalian cation-independent mannose 6-phosphate (Man 6-P) receptor, which mediates trafficking of newly synthesized lysosomal acid hydrolases to lysosomes. However, flies lack the enzymes necessary to make the Man 6-P mark, and the amino acids implicated in Man 6-P binding by the mammalian receptor are not conserved in LERP. Thus, the function of LERP in sorting of lysosomal enzymes to lysosomes in Drosophila is unclear. Here, we analyze the consequence of LERP depletion in S2 cells and intact flies. RNAi-mediated knockdown of LERP in S2 cells had little or no effect on the cellular content or secretion of several lysosomal hydrolases. We generated a novel Lerp null mutation, Lerp(F6), which abolishes LERP protein expression. Lerp mutants have normal viability and fertility and display no overt phenotypes other than reduced body weight. Lerp mutant flies exhibit a 30-40% decrease in the level of several lysosomal hydrolases, and are hypersensitive to dietary chloroquine and starvation, consistent with impaired lysosome function. Loss of LERP also enhances an eye phenotype associated with defective autophagy. Our findings implicate Lerp in lysosome function and autophagy.
    Preview · Article · Sep 2015 · Biology Open
  • Jaime J Barea · Eline van Meel · Stuart Kornfeld · Lynne M Bird

    No preview · Article · Jun 2015 · American Journal of Medical Genetics Part A
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    ABSTRACT: Mucolipidosis II and III alpha/beta (ML II/III alpha/beta) are rare autosomal recessive lysosomal storage diseases that are caused by a deficiency of UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase, the enzyme responsible for the synthesis of the mannose 6-phosphate targeting signal on lysosomal hydrolases. A Brazilian patient suspected of having a very mild ML III was investigated using whole next-generation sequencing (NGS). Two mutations in the GNPTAB gene were detected and confirmed to be in trans status by parental analysis: c.1208T>C (p.Ile403Thr), previously reported as being pathogenic, and the novel mutation c.1723G>A (p.Gly575Arg). This study demonstrates the effectiveness of using whole NGS for the molecular diagnosis of very mild ML III alpha/beta patients.
    Full-text · Article · Mar 2015 · Molecular Genetics and Metabolism Reports
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    ABSTRACT: UDP-GlcNAc:lysosomal enzyme GlcNAc-1-phosphotransferase tags newly synthesized lysosomal enzymes with mannose 6-phosphate recognition markers, which are required for their targeting to the endo-lysosomal system. GNPTAB encodes the α and β subunits of GlcNAc-1-phosphotransferase and mutations in this gene cause the lysosomal storage disorders mucolipidosis II and III αβ. Prior investigation of missense mutations in GNPTAB uncovered amino acids in the N-terminal region and within the DMAP domain involved in Golgi retention of GlcNAc-1-phosphotransferase and its ability to specifically recognize lysosomal hydrolases, respectively. Here, we undertook a comprehensive analysis of the remaining missense mutations in GNPTAB reported in mucolipidosis II and III αβ patients using cell- and zebrafish-based approaches. We show that the Stealth domain harbors the catalytic site, since some mutations in these regions greatly impaired the activity of the enzyme without affecting its Golgi localization and proteolytic processing. We also demonstrate a role for the Notch repeat 1 in lysosomal hydrolase recognition, since missense mutations in conserved cysteine residues in this domain do not affect the catalytic activity, but impair mannose phosphorylation of certain lysosomal hydrolases. Rescue experiments using mRNA bearing Notch repeat 1 mutations in GNPTAB-deficient zebrafish revealed selective effects on hydrolase recognition that differ from the DMAP mutation. Finally, the mutant R587P, located in the spacer between Notch 2 and DMAP, was partially rescued by overexpression of the γ subunit, suggesting a role for this region in γ subunit binding. These studies provide new insight into the functions of the different domains of the α and β subunits. Copyright © 2014, The American Society for Biochemistry and Molecular Biology.
    No preview · Article · Dec 2014 · Journal of Biological Chemistry
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    ABSTRACT: UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase is an α2β2γ2 hexameric enzyme that catalyzes the synthesis of the mannose 6-phosphate targeting signal on lysosomal hydrolases. Mutations in the α/β subunit precursor gene cause the severe lysosomal storage disorder mucolipidosis II (ML II) or the more moderate mucolipidosis III alpha/beta (ML III α/β), while mutations in the γ subunit gene cause the mildest disorder, mucolipidosis III gamma (ML III γ). Here we report neurologic consequences of mouse models of ML II and ML III γ. The ML II mice have a total loss of acid hydrolase phosphorylation, which results in depletion of acid hydrolases in mesenchymal-derived cells. The ML III γ mice retain partial phosphorylation. However, in both cases, total brain extracts have normal or near normal activity of many acid hydrolases reflecting mannose 6-phosphate-independent lysosomal targeting pathways. While behavioral deficits occur in both models, the onset of these changes occurs sooner and the severity is greater in the ML II mice. The ML II mice undergo progressive neurodegeneration with neuronal loss, astrocytosis, microgliosis and Purkinje cell depletion which was evident at 4 months whereas ML III γ mice have only mild to moderate astrocytosis and microgliosis at 12 months. Both models accumulate the ganglioside GM2, but only ML II mice accumulate fucosylated glycans. We conclude that in spite of active mannose 6-phosphate-independent targeting pathways in the brain, there are cell types that require at least partial phosphorylation function to avoid lysosomal dysfunction and the associated neurodegeneration and behavioral impairments.
    Full-text · Article · Oct 2014 · PLoS ONE
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    Balraj Doray · Jennifer Govero · Stuart Kornfeld
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    ABSTRACT: The functional redundancy of the three mammalian GGAs (Golgi-localized, γ-ear containing, ADP-ribosylation factor- binding proteins) was addressed in a previous study. Using insertional mutagenesis, we found that Gga1 or Gga3 homozygous knockout mice were for the most part normal, whereas mice homozygous for two different Gga2 gene-trap alleles exhibited either embryonic or neonatal lethality in the C57BL/6 background, depending on the source of the vector utilized (Byg versus Tigm, respectively). We now show that the Byg strain harbors a disrupted Gga2 allele that is hypomorphic, indicating that the Byg lethality is due to a mechanism independent of GGA2. This is in contrast to the Tigm Gga2 allele, which is a true knockout and establishes a role for GGA2 during the neonatal period. Placement of the Tigm Gga2 allele into the C57BL6/Ola129Sv mixed background results in a lower incidence of neonatal lethality, showing the importance of genetic background in determining the requirement for GGA2 during this period. The Gga2(-/-) mice that survive have a reduced body weight at birth and this runted phenotype is maintained through adulthood.
    Preview · Article · Mar 2014 · G3-Genes Genomes Genetics
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    Eline van Meel · Yi Qian · Stuart A Kornfeld
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    ABSTRACT: The lysosomal storage disorder mucolipidosis III αβ is caused by mutations in the αβ subunits of UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (phosphotransferase). This Golgi-localized enzyme mediates the first step in the synthesis of the mannose 6-phosphate recognition marker on lysosomal acid hydrolases, and loss of function results in impaired lysosomal targeting of these acid hydrolases and decreased lysosomal degradation. Here we show that two patient missense mutations, Lys4Gln and Ser15Tyr, in the N-terminal cytoplasmic tail of the α subunit of phosphotransferase impair retention of the catalytically active enzyme in the Golgi complex. This results in mistargeting of the mutant phosphotransferases to lysosomes, where they are degraded, or to the cell surface and release into the medium. The finding that mislocalization of active phosphotransferase is the basis for mucolipidosis III αβ in a subset of patients shows the importance of single residues in the cytoplasmic tail of a Golgi-resident protein for localization to this compartment.
    Preview · Article · Feb 2014 · Proceedings of the National Academy of Sciences

  • No preview · Article · Feb 2014 · Molecular Genetics and Metabolism
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    ABSTRACT: Methionyl-tRNA synthetase (MARS) catalyzes the ligation of methionine to its cognate transfer RNA and therefore plays an essential role in protein biosynthesis. We used exome sequencing, aminoacylation assays, homology modeling, and immune-isolation of transfected MARS to identify and characterize mutations in the methionyl-tRNA synthetase gene (MARS) in an infant with an unexplained multi-organ phenotype. We identified compound heterozygous mutations (F370L and I523T) in highly conserved regions of MARS. The parents were each heterozygous for one of the mutations. Aminoacylation assays documented that the F370L and I523T MARS mutants had 18 +/- 6% and 16 +/- 6%, respectively, of wild-type activity. Homology modeling of the human MARS sequence with the structure of E. coli MARS showed that the F370L and I523T mutations are in close proximity to each other, with residue I523 located in the methionine binding pocket. We found that the F370L and I523T mutations did not affect the association of MARS with the multisynthetase complex. This infant expands the catalogue of inherited human diseases caused by mutations in aminoacyl-tRNA synthetase genes.
    Full-text · Article · Oct 2013 · BMC Medical Genetics
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    ABSTRACT: UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase) is an α2β2γ2 heterohexamer that mediates the initial step in the formation of the mannose 6-phosphate recognition signal on lysosomal acid hydrolases. We previously reported that the specificity of the reaction is determined by the ability of the α/β subunits to recognize a conformation-dependent protein determinant present on the acid hydrolases. We now present evidence that the DNA methyltransferase-associated protein (DMAP) interaction domain of the α subunit functions in this recognition process. First, GST-DMAP pulled down several acid hydrolases, but not nonlysosomal glycoproteins. Second, recombinant GlcNAc-1-phosphotransferase containing a missense mutation in the DMAP interaction domain (Lys732Asn) identified in a patient with mucolipidosis II exhibited full activity toward the simple sugar α-methyl d-mannoside but impaired phosphorylation of acid hydrolases. Finally, unlike the WT enzyme, expression of the K732N mutant in a zebrafish model of mucolipidosis II failed to correct the phenotypic abnormalities. These results indicate that the DMAP interaction domain of the α subunit functions in the selective recognition of acid hydrolase substrates and provides an explanation for the impaired phosphorylation of acid hydrolases in a patient with mucolipidosis II.
    Preview · Article · Jun 2013 · Proceedings of the National Academy of Sciences
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    ABSTRACT: DUF2233, a Domain of Unknown Function (DUF), is present in many bacterial and several viral proteins, and was also identified in the mammalian transmembrane glycoprotein α-N-acetylglucosamine-1-phosphodiester N-acetylglucosaminidase ("Uncovering enzyme", UCE). We report the crystal structure of BACOVA_00430, a 315-residue protein from the human gut bacterium Bacteroides ovatus, which is the first structural representative of the DUF2233 protein family. A notable feature of this structure is the presence of a surface cavity that is populated by residues that are highly conserved across the entire family. The crystal structure was used to model the luminal portion of human UCE (hUCE), which is involved in targeting of lysosomal enzymes. Mutational analysis of several residues in a highly conserved surface cavity of hUCE revealed that they are essential for function. The bacterial enzyme (BACOVA_00430) has approximately 1% of the catalytic activity of hUCE toward the substrate GlcNAc-P-mannose, the precursor of the Man-6-P lysosomal targeting signal. GlcNAc-1-P is a poor substrate for both enzymes. We conclude that, for at least a subset of proteins in this family, DUF2233 functions as a phosphodiester glycosidase.
    No preview · Article · Apr 2013 · Journal of Biological Chemistry
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    Balraj Doray · Saurav Misra · Yi Qian · Tom J Brett · Stuart Kornfeld
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    ABSTRACT: The GGA family of clathrin adaptor proteins mediates the intracellular trafficking of transmembrane proteins by interacting with DXXLL-type sorting signals on the latter. These signals were originally identified at the carboxy-termini of the transmembrane cargo proteins. Subsequent studies, however, showed that internal DXXLL sorting motifs occur within the N- or C-terminal cytoplasmic domains of cargo molecules. The GGAs themselves also contain internal DXXLL motifs that serve to auto-regulate GGA function. A recent study challenged the notion that internal DXXLL signals are competent for binding to GGAs. Since the question of whether GGA adaptors interact with internal DXXLL motifs is fundamental to the identification of bona fide GGA cargo, and to an accurate understanding of GGA regulation within cells, we have extended our previous findings. We now present additional evidence confirming that GGAs do interact with internal DXXLL motifs. We also summarize the recent reports from other laboratories documenting internal GGA binding motifs.
    Full-text · Article · Jul 2012 · Traffic
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    Jennifer Govero · Balraj Doray · Hongdong Bai · Stuart Kornfeld
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    ABSTRACT: Numerous studies using cultured mammalian cells have shown that the three GGAs (Golgi-localized, gamma-ear containing, ADP-ribosylation factor- binding proteins) function in the transport of cargo proteins between the trans- Golgi network and endosomes. However, the in vivo role(s) of these adaptor proteins and their possible functional redundancy has not been analyzed. In this study, the genes encoding GGAs1-3 were disrupted in mice by insertional mutagenesis. Loss of GGA1 or GGA3 alone was well tolerated whereas the absence of GGA2 resulted in embryonic or neonatal lethality, depending on the genetic background of the mice. Thus, GGA2 mediates a vital function that cannot be compensated for by GGA1and/or GGA3. The combined loss of GGA1 and GGA3 also resulted in a high incidence of neonatal mortality but in this case the expression level of GGA2 may be inadequate to compensate for the loss of the other two GGAs. We conclude that the three mammalian GGAs are essential proteins that are not fully redundant.
    Preview · Article · Jan 2012 · PLoS ONE
  • Wang-Sik Lee · Changsoo Kang · Dennis Drayna · Stuart Kornfeld
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    ABSTRACT: GlcNAc-1-phosphodiester-N-acetylglucosaminidase ("uncovering enzyme" (UCE); EC 3.1.4.45) is a Golgi enzyme that mediates the second step in the synthesis of the mannose 6-phosphate lysosomal targeting signal on acid hydrolases. Recently, three mutations (two missense and one deletion/frameshift) in the NAGPA gene that encodes UCE have been identified in individuals with persistent stuttering. We now demonstrate that each mutation leads to lower cellular UCE activity. The p.R328C mutation impairs folding in the endoplasmic reticulum, resulting in degradation of a significant portion by the proteasomal system. The p.H84Q mutation also impairs folding and, in addition, decreases the specific activity of the enzyme that folds sufficiently to traffic to the Golgi. The p.F513SfsX113 frameshift mutation adds 113 amino acids to the C terminus of the cytoplasmic tail of the protein, including a VWLL sequence that causes rapid degradation via the proteasomal system. These biochemical findings extend the genetic data implicating mutations in the NAGPA gene in the persistent stuttering phenotype.
    No preview · Article · Sep 2011 · Journal of Biological Chemistry
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    ABSTRACT: Osteoclasts are specialized cells that secrete lysosomal acid hydrolases at the site of bone resorption, a process critical for skeletal formation and remodeling. However, the cellular mechanism underlying this secretion and the organization of the endo-lysosomal system of osteoclasts have remained unclear. We report that osteoclasts differentiated in vitro from murine bone marrow macrophages contain two types of lysosomes. The major species is a secretory lysosome containing cathepsin K and tartrate-resistant acid phosphatase (TRAP), two hydrolases critical for bone resorption. These secretory lysosomes are shown to fuse with the plasma membrane, allowing the regulated release of acid hydrolases at the site of bone resorption. The other type of lysosome contains cathepsin D, but little cathepsin K or TRAP. Osteoclasts from Gnptab(-/-) (gene encoding GlcNAc-1-phosphotransferase α, β-subunits) mice, which lack a functional mannose 6-phosphate (Man-6-P) targeting pathway, show increased secretion of cathepsin K and TRAP and impaired secretory lysosome formation. However, cathepsin D targeting was intact, showing that osteoclasts have a Man-6-P-independent pathway for selected acid hydrolases.
    Full-text · Article · Apr 2011 · Traffic
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    ABSTRACT: We previously reported that mice deficient in UDP-GlcNAc:lysosomal enzyme GlcNAc-1-phosphotransferase (mucolipidosis type II or Gnptab -/- mice), the enzyme that initiates the addition of the mannose 6-phosphate lysosomal sorting signal on acid hydrolases, exhibited extensive vacuolization of their exocrine gland cells, while the liver, brain, and muscle appeared grossly unaffected. Similar pathological findings were observed in several exocrine glands of patients with mucolipidosis II. To understand the basis for this cell type-specific abnormality, we analyzed these tissues in Gnptab -/- mice using a combined immunoelectron microscopy and biochemical approach. We demonstrate that the vacuoles in the exocrine glands are enlarged autolysosomes containing undigested cytoplasmic material that accumulate secondary to deficient lysosomal function. Surprisingly, the acid hydrolase levels in these tissues ranged from normal to modestly decreased, in contrast to skin fibroblasts, which accumulate enlarged lysosomes and/or autolysosomes also but exhibit very low levels of acid hydrolases. We propose that the lysosomal defect in the exocrine cells is caused by the combination of increased secretion of the acid hydrolases via the constitutive pathway along with their entrapment in secretory granules. Taken together, our results provide new insights into the mechanisms of the tissue-specific abnormalities seen in mucolipidosis type II.
    Preview · Article · Feb 2011 · Molecular biology of the cell
  • Stuart Kornfeld
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    ABSTRACT: We now recognize that a large number of membrane and soluble proteins contain covalently linked oligosaccharides that exhibit a vast array of structures and participate in a wide variety of biological processes. Nowhere is this better illustrated than the mannose 6-phosphate (Man-6-P) recognition system that mediates the trafficking of newly synthesized acid hydrolases to lysosomes in higher eukaryotes. The Asn-linked high-mannose oligosaccharides of these hydrolases facilitate folding of the nascent proteins in the endoplasmic reticulum via interaction with lectin-type chaperones and after phosphorylation in the Golgi, function as ligands for binding to Man-6-P receptors, a critical step in their transport to lysosomes. Failure to synthesize the Man-6-P recognition marker results in a serious lysosomal storage disease, one of a growing number of genetic conditions, termed congenital disorders of glycosylation, that result from faulty glycan biosynthesis.
    No preview · Article · Nov 2010 · Molecular biology of the cell
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    Stuart Kornfeld

    Preview · Article · Jul 2010 · The Journal of clinical investigation
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    Yi Qian · Christopher M West · Stuart Kornfeld
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    ABSTRACT: The Dictyostelium discoideum gene gpt1 encodes a protein XP_638036 with sequence similarity to the alpha/beta subunits of mammalian UDP-GlcNAc:Glycoprotein N-acetylglucosamine-1-phosphotransferase. We now demonstrate that extracts of D. discoideum clones with mutations in this gene transfer GlcNAc-P from UDP-GlcNAc to mannose residues at less than 5% the wild type value. Further, the lysosomal hydrolases of these mutant clones fail to bind to a cation-independent mannose 6-phosphate receptor affinity column, indicating a lack of methylphosphomannosyl residues on the high mannose oligosaccharides of these proteins. We conclude that the gpt1 gene product catalyzes the initial step in the formation of methylphosphomannosyl residues on D. discoideum lysosomal hydrolases.
    Preview · Article · Feb 2010 · Biochemical and Biophysical Research Communications

Publication Stats

19k Citations
1,524.09 Total Impact Points

Institutions

  • 1969-2015
    • Washington University in St. Louis
      • • Department of Pediatrics
      • • Department of Medicine
      • • Division of Hematology and oncology
      San Luis, Missouri, United States
  • 1989
    • Medical College of Wisconsin
      • Department of Biochemistry
      Milwaukee, Wisconsin, United States