Tatyana Leonova

University of Cincinnati, Cincinnati, OH, United States

Are you Tatyana Leonova?

Claim your profile

Publications (9)30.91 Total impact

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The reversibility and regression of histological and biochemical findings in a mouse model of Gaucher disease (4L/PS-NA) was evaluated using a liver-enriched activator protein promoter control of a tetracycline-controlled transcriptional activation-responsive human acid beta-glucosidase (hGCase) transgenic system. 4L/PS-NA has the acid beta-glucosidase (GCase) V394L/V394L (4L) point mutation combined with hypomorphic ( approximately 6% wild-type) expression of the mouse prosaposin transgene (PS-NA). The hGCase/4L/PS-NA had exclusive liver expression of hGCase controlled by doxycycline (DOX). In the absence of DOX, hGCase was secreted from liver at levels of approximately 120 microg/ml serum with only approximately 8% of full activity, following exposure to pH 7.4 in serum. The hGCase activity and protein were detected in cells of the liver (massive), lung, and spleen, but not the brain. The visceral tissue storage cells and glucosylceramide (GC) accumulation in hGCase/4L/PS-NA were decreased from that in 4L/PS-NA mice. Turning off hGCase expression with dietary DOX led to reaccumulation of storage cells and of GC in liver, lung, and spleen, and macrophage activation in those tissues. This study demonstrates that conditionally expressed hGCase supplemented the existing mutant mouse GCase to control visceral substrate accumulation in vivo.
    The Journal of Lipid Research 11/2006; 47(10):2161-70. · 4.39 Impact Factor
  • You Hai Xu, Tatyana Leonova, Gregory A Grabowski
    [Show abstract] [Hide abstract]
    ABSTRACT: TCP80 is an approximately 80kDa mammalian cytoplasmic protein that binds to a set of mRNAs and inhibits their translation in vitro and ex vivo. This protein has high sequence similarity to interleukin-2 enhancer-binding factors (NF90/ILF3) and the M-phase phosphoprotein (MPP4)/DRBP76. A 110kDa immunologic isoform of TCP80/NF90/MPP4/DRBP76, termed TCP110, also is present in cytoplasm and nuclei of many types of cells. A cDNA sequence coding for TCP110 was derived by 5(')RACE. The TCP110 sequence is identical to ILF3. The gene coding for TCP110/ILF3 mapped to human chromosome 19 and the gene organization was analyzed using TCP80 and TCP110/ILF3 cDNA sequences. The TCP/ILF3 gene spans >34.8kb and contains 21 exons. At least one alternatively spliced product involving exons 19-21 exists and predicts the formation of either TCP80 or TCP110/ILF3. However, the functional relationships of TCP80 and TCP110/ILF3 required elucidation. The metabolic turnover rates and subcellular distribution of TCP80 and TCP110/ILF3 during the cell cycle showed TCP80 to be relatively stable (t(1/2)=5 days) in the cytoplasmic compartment. In comparison, TCP110/ILF3 migrated between the cytoplasmic and nuclear compartments during the cell cycle. The TCP110 C-terminal segment contains an additional nuclear localizing signal that plays a role in its nuclear translocation. This study indicates that the multiple cellular functions, i.e., translation control, interleukin-2 enhancer binding, or cell division, of TCP/ILF3 are fulfilled by alternatively spliced isoforms.
    Molecular Genetics and Metabolism 12/2003; 80(4):426-36. · 2.83 Impact Factor
  • Tatyana Leonova, Gregory A. Grabowski
    [Show abstract] [Hide abstract]
    ABSTRACT: Gaucher disease (GD) is associated with mutations at the acid β-glucosidase (GCase) locus and the resultant defective activity of the enzyme product. GCase is a membrane-associated glycoprotein that requires detergents for extraction and phospholipid interfaces for full catalytic activity. Normal human fibroblasts and overexpressing transgenic cell lines were used to evaluate the intracellular disappearance, degradation, and secretion of human GCase, including GD fibroblasts and C2C12 cells transduced with MFG-GCase retrovirus and CHO cells stably transfected with the tetracycline transactivation conditional expression system (tet-CHO-GCase). Compared to HF, the disappearance of GCase from the transgenic cells was 12–30 times greater, and had degradative and secretory components. In tet-CHO-GCase cells the majority of GCase was secreted. Intracellular degradation occurred in compartments sensitive to monensin and brefeldin A, and the ALLN or leupeptin protease inhibitors, i.e., ER, Golgi, and lysosomes. In tet-CHO-GCase cells, GCase degradation and secretion rates were inversely related to expression level. Saponin permeabilization analyses of tet-CHO-GCase cells showed that a majority of GCase was soluble, with a rapid disappearance via secretion and degradation. A progressively increasing proportion of GCase became saponin insoluble with a t1/2 = 2–3 h. Intracellular saponin-soluble and -insoluble GCases were degraded with t1/2 ∼2 and 14 h, respectively. Confocal microscopy showed colocalization of glycosylated or unglycosylated GCase with LAMP-2, an integral lysosomal membrane protein, to vesicular bodies. These studies show that GCase secretion was N-linked glycosylation dependent, whereas sorting to and membrane attachment in the lysosome were N-linked glycosylation independent.
    Molecular Genetics and Metabolism 09/2000; · 2.83 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Prosaposin is the precursor of four activator proteins, termed saposins A, B, C, and D, that are required for much of glycosphingolipid hydrolysis. The intact precursor also has neurite outgrowth activity ex vivo and in vivo that is localized to amino acid residues 22−31 of saposin C. Across species, this saposin C region has a high degree of identity and similarity with amino acids in the analogous region of saposin A. Wild-type and mutant saposins C and A from human and mouse were expressed in E. coli. Pure proteins, synthetic peptide analogues, conformation-specific antibodies, and CD spectroscopy were used to evaluate the basis of the ex vivo neuritogenic effect. Wild-type saposin A had no neuritogenic activity whereas reduced and alkylated saposin A did. Introduction of the conserved saposin A Tyr 30 (Y30) into saposin C at the analogous position 31, a conserved Ala(A)/Gly(G)31, diminished neuritogenic activity by 50−60%. Nondenatured saposin A with an introduced A30 acquired substantial neuritogenic activity. Polyclonal antibodies directed against the NH2-terminus of saposin C cross-reacted well with reduced and alkylated saposins C and A, wild-type saposin C, and saposin A [Y30A], poorly with saposin C [A31Y], and not at all with wild-type saposin A. CD spectra of wild-type and mutant saposins C and A, the corresponding neuritogenic region of saposin C, and the analogous region of saposin A showed that more “saposin C-like” molecules had neuritogenic properties. Those with more “saposin A-like” spectra did not. These studies show that the neuritogenic activity of saposin C requires specific placement of amino acids, and that Y30 of saposin A significantly alters local conformation in this critical region and suppresses neuritogenic activity.
    Biochemistry 04/1999; 38(19). · 3.38 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Human lysosomal acid lipase (hLAL) is essential for the hydrolysis of cholesteryl esters and triglycerides in the lysosome. Defective hLAL activity leads to two autosomal recessive traits, Wolman disease (WD) or cholesteryl ester storage disease (CESD). Phenotypically, WD has accumulation of both triglycerides and cholesteryl esters, while CESD has mainly elevated cholesteryl esters. We characterized mutations in the hLAL gene from two CESD siblings. By reverse transcriptase-PCR (RT-PCR) and cDNA cloning and sequencing, we identified homozygous deletion mutations of nucleotides 863 to 934, in the hLAL transcript. Normal levels of LAL mRNA were detected. The deletion in mRNA is due to a G to A transition in the last nucleotide of exon 8 of the hLAL gene, a splice junction mutation (E8SJM) that resulted in exon skipping, and a predicted in-frame deletion of the 24 amino acids. [35S]Met metabolic labeling studies in fibroblasts showed a low level of E8SJM LAL (∼38%) that was highly unstable. Heterologous expression of E8SJM LAL in insect cells gave an LAL with low catalytic activity toward cholesteryl oleate and triolein. The effects of this mutation are complex with the production of decreased amounts of an unstable LAL that is catalytically defective. The results suggest that E8SJM leads to essentially a null allele and that the differences in WD and CESD phenotype involve other factors.
    Molecular Genetics and Metabolism 07/1998; · 2.83 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Prosaposin is a multifunctional protein encoded at a single locus in humans and mice. The precursor contains, in tandem, four glycoprotein activators or saposins, termed A, B, C, and D, that are essential for specific glycosphingolipid hydrolase activities. Prosaposin appears to be a potent neurotrophic factor. To explore the proteolytic processing from prosaposin to mature activator proteins, metabolic labeling was done with human prosaposin expressed in insect cells, human fibroblasts, neuronal stem cells (NT2) and retinoic acid-differentiated NT2 neurons. In all cell types, the major processing pathway was through a tetrasaposin, A-B-C-D, from which saposin A was then removed. In mammalian cells monosaposins were derived from the trisaposin B-C-D by cleavage to the disaposins, B-C and C-D, that were processed to monosaposins. In insect cells the major end products were the disaposins, with A-B and C-D derived from the tetrasaposin, A-B-C-D, or with B-C and C-D derived from the trisaposin, B-C-D. In insect and mammalian cells, the nonsignal NH2-terminal peptide preceding saposin A (termed Nter) was usually removed prior to saposin A cleavage. In NT2-derived differentiated neurons, precursor tetrasaposins containing A-B-C-D were secreted with and without Nter. Immunofluorescence studies using prosaposin-specific antisera showed large steady state amounts of uncleaved prosaposin in Purkinje cells, cortical neurons, and other specific cell types in adult mice. These studies indicate that prosaposin processing is highly regulated at a proteolytic level to produce prosaposin, tetrasaposins, or mature monosaposins in specific mammalian cells.
    Journal of Biological Chemistry 07/1996; 271(29):17312-17320. · 4.65 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Gaucher disease type 1, the most prevalent lysosomal storage disease, is caused by the defective activity of the lysosomal enzyme, acid beta-glucosidase, or glucocerebrosidase. Infusion of purified acid beta-glucosidase containing alpha-mannosyl-terminated oligosaccharides (alglucerase) is efficacious in reversing hematologic, hepatic, splenic, and bony disease manifestations. The murine tissue distribution and turnover of bolus injections of alglucerase was evaluated by enzymatic activity, quantitative cross-reacting immunologic material analyses, and immunofluorescence studies. Enzyme activity measurements detected distribution to liver, spleen, thymus, kidney, and bone marrow mononuclear cells, but not to lungs and brain. In kidney and thymus, the enzyme was transiently present. In liver and spleen, enzyme activity peaked at about 20 min postinjection followed by a biphasic decrease with t1/2 approximately 40-60 min and approximately 12-14 h. In bone marrow maximal enzyme activity was at 40-60 min with a disappearance t1/2 approximately 60 min. Quantitative cross-reacting immunologic material studies of liver and spleen showed delivery of enzyme with decreased catalytic rate constants whose degradation included denaturation and proteolytic components. By immunofluorescence the human enzyme was distributed primarily to reticuloendothelial cells of the liver and spleen. In autopsy material from a Gaucher disease type 2 patient treated with enzyme, immunohistochemical and activity studies showed distributions similar to those in mice. These studies indicate a complex delivery and intracellular degradation of acid beta-glucosidase with lower intrinsic activity than the administered therapeutic agent.
    Pediatric Research 03/1996; 39(2):313-22. · 2.67 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Gaucher disease type 1, the most prevalent lysosomal storage disease, is caused by the defective activity of the lysosomal enzyme, acidβ-glucosidase, or glucocerebrosidase. Infusion of purified acidβ-glucosidase containing α-mannosyl-terminated oligosaccharides(alglucerase) is efficacious in reversing hematologic, hepatic, splenic, and bony disease manifestations. The murine tissue distribution and turnover of bolus injections of alglucerase was evaluated by enzymatic activity, quantitative cross-reacting immunologic material analyses, and immunofluorescence studies. Enzyme activity measurements detected distribution to liver, spleen, thymus, kidney, and bone marrow mononuclear cells, but not to lungs and brain. In kidney and thymus, the enzyme was transiently present. In liver and spleen, enzyme activity peaked at about 20 min postinjection followed by a biphasic decrease with t½ ≈ 40-60 min and≈ 12-14 h. In bone marrow maximal enzyme activity was at 40-60 min with a disappearance t½ ≈ 60 min. Quantitative cross-reacting immunologic material studies of liver and spleen showed delivery of enzyme with decreased catalytic rate constants whose degradation included denaturation and proteolytic components. By immunofluorescence the human enzyme was distributed primarily to reticuloendothelial cells of the liver and spleen. In autopsy material from a Gaucher disease type 2 patient treated with enzyme, immunohistochemical and activity studies showed distributions similar to those in mice. These studies indicate a complex delivery and intracellular degradation of acid β-glucosidase with lower intrinsic activity than the administered therapeutic agent.
    Pediatric Research 01/1996; 39(2):313-322. · 2.67 Impact Factor
  • Source
    X Qi, T Leonova, G A Grabowski
    [Show abstract] [Hide abstract]
    ABSTRACT: Small (80-amino acid) glycoproteins or saposins are important for the in vivo function of several lysosomal hydrolases. Four saposins, A, B, C, and D, are encoded by a single locus termed prosaposin. Saposins C and A are thought to function in vivo as activators of acid beta-glucosidase. The physiologic role of saposin C has been confirmed, whereas that of saposin A role has not. To investigate the effects of saposins C and A on acid beta-glucosidase activity, the coding sequence for the individual saposins was expressed in Escherichia coli and the recombinant proteins purified to homogeneity. Recombinant and natural saposins A and C activated acid beta-glucosidase similarly only in micromolar amounts. Saposin C had specific activation of acid beta-glucosidase activity at < 200 nM. A second phase of activation was achieved at > 1 microM. In comparison, saposin A consistently activated acid beta-glucosidase only at > 1 microM. Two mutant saposins C (Cys382-->Phe and Cys382--Gly) were created and shown to compete with saposin C for a site on acid beta-glucosidase. The mutant saposins did not activate the enzyme. Recombinant saposin A (< 200 nM) competed with saposin C for a site on the enzyme but without activating effects. These studies show that saposin A is not an in vitro activator of acid beta-glucosidase at physiologic concentrations, although binding occurs without activating acid beta-glucosidase. The studies with mutant saposins C indicate that the binding and activation effects of saposins C are distinct events. These results indicate that the saposin C-induced conformational change in the enzyme occurs via highly specific, probably multivalent, interactions between acid beta-glucosidase and saposin C.
    Journal of Biological Chemistry 07/1994; 269(24):16746-53. · 4.65 Impact Factor

Publication Stats

197 Citations
30.91 Total Impact Points

Institutions

  • 1998–2006
    • University of Cincinnati
      • • Department of Pediatrics
      • • Department of Molecular Genetics, Biochemistry, and Microbiology
      • • College of Medicine
      Cincinnati, OH, United States
  • 2000–2003
    • Cincinnati Children's Hospital Medical Center
      • Division of Human Genetics
      Cincinnati, OH, United States