B12 METABOLISM IN HUMANS By NICOLE AURORA LEAL A ...

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54 Prior studies have indicated that ATR defects in humans lead to methylmalonic aciduria (Fenton and Rosenberg 1981, Banerjee and Chowdhury 1999). MCM is known to require AdoCbl for activity (Banerjee and Chowdhury 1999) and fibroblast extracts from methylmalonic aciduria patients with defects in the cblB complementation group have been shown to lack ATR activity (Fenton and Rosenberg 1981). The ATR cDNA identified in this study corresponded to a single human gene comprised of nine exons found on chromosome XII. Furthermore, Western blots indicated that expression of this ATR was altered in cblB mutant human cell lines compared to normal cell lines. Thus, we propose that the human gene identified here corresponds to the cblB complementation group that is involved in methylmalonic aciduria. The approach used in this study to identify the human ATR cDNA involved screening mammalian cDNA expression libraries for clones that complemented an ATR-deficient bacterial mutant. This general strategy may have application to the identification of additional cDNAs involved in cobalamin metabolism. Human cDNAs encoding the β-ligand transferase and the cob(III)alamin and cob(II)alamin reductases (the cblACD complementation groups) have not yet been identified (Rosenblatt and Fenton 1999, Watkins and Rosenblatt 2001). In this regard, it is notable that in this study the human ATR cDNA was not directly isolated, but rather the bovine ATR cDNA was isolated and sequence similarity searching was used to identify the homologous human gene. The fact that our screen allowed direct isolation of a bovine ATR cDNA, but not a human ATR cDNA may reflect the relative abundance of ATR mRNA in the human versus the bovine liver. Ruminant livers have among the highest levels of <strong>B12</strong>-dependent enzymes measured in any tissue (Schneider and Stroinski 1987). Thus, ruminant liver
libraries may be particularly useful for isolating mammalian cDNAs involved in cobalamin metabolism. 55 Importantly, the identification of human genes involved in methylmalonic aciduria should help with the development of improved methods for diagnosis and treatment of this rare but devastating disease. Knowledge of the relevant genes will allow DNA-based methods of diagnosis following amniocentesis or chorionic villi sampling. Such techniques will allow the identification of the specific genetic lesions involved, and this may help guide treatment. Some cases of methylmalonic aciduria respond to high-dose <strong>B12</strong> therapy and it seems likely that such cases will be associated with specific mutations (Ampola et al. 1975, Matsui et al. 1983, Zass et al. 1995). Moreover, as the needed methodologies become available, knowledge of the genes involved in cobalamin metabolism may also help with the development of gene or enzyme therapies as treatments for methylmalonic aciduria.
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B12 METABOLISM IN HUMANS By NICOLE
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The work in this dissertation is de
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TABLE OF CONTENTS Page ACKNOWLEDGME
Page 7 and 8:
General Molecular and Protein Metho
Page 9 and 10:
LIST OF TABLES Table page 1-1. Aden
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3-7. Stoichiometry of the MSR-ATR s
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DTT dithiothreitol E. coli Escheric
Page 15 and 16:
Abstract of Dissertation Presented
Page 17 and 18:
CHAPTER 1 INTRODUCTION History of C
Page 19 and 20: 3 (DMB). The DMB moiety is covalent
Page 21 and 22: 5 and deoxyadenosine. After hydroge
Page 23 and 24: 7 homocysteine recycling and methio
Page 25 and 26: methylmalonic acid. Methylmalonic a
Page 27 and 28: folate-dependent reactions includin
Page 29 and 30: tetanomorphum, P. shermanii, Euglen
Page 31 and 32: enzyme. This suggests that cob(II)a
Page 33 and 34: at the 5’-C of ATP by cob(I)alami
Page 35 and 36: iochemical studies and complementat
Page 37 and 38: 1997). Both cases of the cblD disor
Page 39 and 40: 23 The cblB complementation group i
Page 41 and 42: (Watkins et al. 2002). Some patient
Page 43 and 44: Chapter 2 of this dissertation repo
Page 45 and 46: Corrin Ring Dimethyl benzimidazole
Page 47 and 48: Classes Eliminases Dehydratases OH
Page 49 and 50: A) B) CH 3THF 33 MS-cob(I)alamin Me
Page 51 and 52: propanol dehydrogenase (PduQ) propa
Page 53 and 54: or from mutations that impair the a
Page 55 and 56: 1989). 5-bromo-4-chloro-3-indolyl-
Page 57 and 58: 41 5’-GCCGCCGGTACCGATGACGACGACAAG
Page 59 and 60: 43 Growth of Adenosyltransferase Ex
Page 61 and 62: anti-rabbit IgG (γ-chain specific)
Page 63 and 64: sequence similarity to a known ATR
Page 65 and 66: ATRs were produced as fusion protei
Page 67 and 68: 51 of the lacI repressor (not shown
Page 69: in which proteins bind B12, the "ba
Page 73 and 74: 57 Figure 2-1. Multiple sequence al
Page 75 and 76: Table 2-2. Specific activities of b
Page 77 and 78: 61 1 2 3 4 5 25 kDa Figure 2-4. Wes
Page 79 and 80: 63 (cyanocobalamin, CNCbl) and hydr
Page 81 and 82: 65 5’- triphosphate (ATP), and di
Page 83 and 84: extracts of ATR 239K (160 mg protei
Page 85 and 86: Milford, MA). Samples (200 µl) wer
Page 87 and 88: Linearity of the ATR reaction 71 Th
Page 89 and 90: 73 reactions was characteristic of
Page 91 and 92: 75 MSR Produces little Cob(I)alamin
Page 93 and 94: Johnson et al. 2001, Saridakis et a
Page 95 and 96: AdoCbl by the MSR-ATR system. Exper
Page 97 and 98: propionyl-CoA PCC (2S)-methylmalony
Page 99 and 100: kDa 97 66 45 31 21 14 7 83 1 2 3 4
Page 101 and 102: 85 Table 3-3. The MSR-ATR system se
Page 103 and 104: Absorbance 0.5 0.4 0.3 0.2 0.1 0.0
Page 105 and 106: Wavelength, (525 nm) 0.24 0.23 0.22
Page 107 and 108: Activity, (nmol min-1 Activity, (nm
Page 109 and 110: al. 1988). The aldehyde is then dis
Page 111 and 112: CoA-acylating propionaldehyde dehyd
Page 113 and 114: 97 previously published DNA sequenc
Page 115 and 116: mM CHES (2-[N-cyclohexylamino]ethan
Page 117 and 118: 101 For the conjugation step, strai
Page 119 and 120: 103 from New England Biolabs (Bever
Page 121 and 122:
105 Propionaldehyde Dehydrogenase A
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107 fraction. Following centrifugat
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109 antiserum obtained was specific
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111 biochemical evidence was presen
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113 Table 4-1. Bacterial strains Sp
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115 Table 4-3. Propionaldehyde dehy
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kDa 209 80 49 35 29 117 1 2 3 4 Fig
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CHAPTER 5 CONCLUSIONS In humans, co
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121 substitutions that are common p
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123 Studies also indicated that hum
Page 141 and 142:
LIST OF REFERENCES Aberg, A., S. Ha
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127 Bradford, M. 1976. A rapid and
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129 Fenton, W. A. and L. E. Rosenbe
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131 Johnson, C. L. V. J., E. Pechon
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133 Mellman, I., H. F. Willard and
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135 Rossi, M., J. P. Glusker, L. Ra
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137 Vlasie, M., S. Chowdhury and R.
Page 155 and 156:
BIOGRAPHICAL SKETCH Nicole Aurora L
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