My research program involves molecular biological approaches to studying inherited metabolic disorders. My current major focus is alanine:glyoxylate aminotransferase (AGT ), a liver peroxisomal enzyme. AGT deficiency causes primary hyperoxaluria type 1 (PH1), an inherited kidney disease primarily affecting children, causing calcium oxalate stones and progressive kidney failure.

Two striking features of PH1 are: 1) In the most common form of PH1, AGT is active but is incorrectly targeted to the mitochondria, where it is physically separated from its substrate located in peroxisomes; 2) About 50% of PH1 patients respond positively to vitamin B6 therapy. This effect is usually associated with the mis-targeting defect but its mechanism is not fully understood.

I am investigating the basis of the response to B6 therapy, the effects of selected mutations on the ability of AGT to form a functional enzyme, and the potential for agents like vitamin B6 to restore enzyme activity in mutant AGT proteins.

My present research focuses on primary hyperoxaluria type 1 (PH1), a severe genetic kidney disease caused by a deficiency of alanine:glyoxylate aminotransferase (AGT), a liver peroxisomal enzyme. This research progresses on three fronts:  1. The identification of mutations and polymorphisms associated with PH1 and their application to diagnosis; 2. The diverse consequences of missense mutations; and 3. The potential for therapeutic intervention.

Specific projects:

1. Identification of mutations and polymorphisms in the AGT gene

More than 140 mutations are now documented in the AGT gene; about 20% have been identified in my laboratory. About 70% of PH1 patients will have at least one of four common mutations. Missense mutations comprise about 40% of the known AGT mutations. These are enrolled in the second area of my research; i.e., investigation of the consequences of missense.

2. Consequences of missense mutations and the potential for intervention

Missense rarely inactivates an enzyme directly but rather leads to mis-folding, which in turn leads to reduced affinity for co-factor, aberrant dimerization, aggregation, and increased susceptibility to proteasome-mediated degradation. The consequence is loss of enzyme activity by an otherwise potentially intact enzyme. I am using three in vitro expression systems in which I can isolate various aspects of AGT synthesis and biogenesis in normal and mutant proteins and begin to reconstruct events in vivo : overexpression of human AGT in E. coli, in vitro transcription/translation with rabbit reticulocyte lysates, and transient expression in tissue culture cells. Combinations of these methods are being used in the following areas:

Cofactor affinity : We already know that pyridoxine therapy decreases oxalate levels in a subset of PH1 patients. Our work has shown that this may be due, in part, to a decreased affinity of the mutant AGT for pyridoxal phosphate (PLP). We are investigating the effects on PLP affinity of a series of missense mutants.

Dimerization and aggregation of AGT: Delayed dimerization is a critical factor in the mis-targeting of AGT to mitochondria in the most common phenotype. We are investigating the potential for PLP to alter the rate of dimerization and decrease nonspecific aggregation.

Stability of mutant protein to quality control surveillance: Mis-folded proteins are frequently targets of ubiquitination and proteasome-mediated degradation in a process of quality control. This phenomenon has been well-documented for proteins that enter the endoplasmic reticulum. Peroxisomal proteins such as AGT have received less attention. We are investigating the role of ubiquitination and the proteasome in the degradation of various mutant of AGT.

3. Potential for therapeutic intervention with missense mutations of AGT:
Potential for chemical “chaperone” effects on mutant stability and enzymatic activity: We have shown enhanced stability for mutant proteins in the presence of PLP and the substrate analog, amino oxyacetic acid.  We are investigating additional small molecules for their potential ability to prevent degradation of the mutant enzyme and to rescue enzymatic activity.

Coulter-Mackie MB, 4-Hydroxyproline metabolism and glyoxylate production: a target for substrate depletion in primary hyperoxaluria type? Kidney Int 2006; 70:1891-1893.

Coulter-Mackie MB, Lian Q,  Consequences of missense mutations for dimerization and turnover of alanine:glyoxylate aminotransferase: Study of a spectrum of mutations. Mol Genet Metab 2006;  89:349-359.

Coulter-Mackie MB , White CT , Hurley RM, Primary hyperoxaluria type 1. Gene Reviews: Genetic Disease Online Reviews 2006. (http://www.geneclinics.org )

Coulter-Mackie MB, Lian Q, Applegarth D, Toone J,  The major allele of the alanine:glyoxylate aminotransferase gene: nine novel mutations and polymorphisms associated with primary hyperoxaluria type 1. Mol Genet Metab 2005; 86: 172-178.

Coulter-Mackie MB,  Preliminary evidence for ethnic differences in primary hyperoxaluria type 1 genotype. Am J Nephrol 2005; 25: 264-268.

Coulter-Mackie MB, Lian Q, Wong S, Overexpression of human alanine:glyoxylate aminotransferase in E. coli: renaturation from guanidine-HCl and affinity for pyridoxal phosphate co-factor. Prot Exp Purif 2005; 41: 18-26.

Rumsby G, Williams E, Coulter-Mackie MB, Evaluation of mutation screening as a first line test for the diagnosis of the primary hyperoxalurias. Kidney Int 2004; 66: 959-963.

Coulter-Mackie MB, Applegarth D, Toone JR, Henderson H, The major allele of the alanine:glyoxylate aminotransferase gene: seven novel mutations causing primary hyperoxaluria type 1. Mol.Genet Metab 2004; 82: 64-68.