Primary hyperoxaluria type 1 in the Canary Islands: a conformational disease due to I244T mutation in the P11L-containing alanine:glyoxylate aminotransferase

Abstract

Primary hyperoxaluria type 1 (PH1) is an inborn error of metabolism resulting from a deficiency of alanine:glyoxylate aminotransferase (AGXT; EC 2.6.1.44). Most of the PH1 alleles detected in the Canary Islands carry the Ile-244 --> Thr (I244T) mutation in the AGXT gene, with 14 of 16 patients homozygous for this mutation. Four polymorphisms within AGXT and regional microsatellites also were shared in their haplotypes (AGXT*LTM), consistent with a founder effect. The consequences of these amino acid changes were investigated. Although I244T alone did not affect AGXT activity or subcellular localization, when present in the same protein molecule as Leu-11 --> Pro (L11P), it resulted in loss of enzymatic activity in soluble cell extracts. Like its normal counterpart, the AGXT*LTM protein was present in the peroxisomes but it was insoluble in detergent-free buffers. The polymorphism L11P behaved as an intragenic modifier of the I244T mutation, with the resulting protein undergoing stable interaction with molecular chaperones and aggregation. This aggregation was temperature-sensitive. AGXT*LTM expressed in Escherichia coli, as a GST-fusion protein, and in insect cells could be purified and retained enzymatic activity. Among various chemical chaperones tested in cell culture, betaine substantially improved the solubility of the mutant protein and the enzymatic activity in cell lysates. In summary, I244T, the second most common mutation responsible for PH1, is a protein conformational disease that may benefit from new therapies with pharmacological chaperones or small molecules to minimize protein aggregation.

Figures

Fig. 1.

Confocal microscopy of COS7 cells transfected with Flag-tagged plasmids expressing either the wild-type AGXT or the AGXT*LTM cDNA. Mouse anti-Flag antibodies and FITC-labeled anti-mouse IgG were used to detect the expressed AGXT protein. The peroxisomes were identified in the same cells with rabbit anti-PMP70 serum and rhodamine-labeled anti-rabbit IgG. The mitochondria were labeled with red fluorescent Mitotracker. Both wild-type AGXT and AGXT*LTM proteins show a peroxisomal distribution, with the merged images (Center) producing yellow fluorescence because of the colocalization of AGXT and PMP70. No mitochondrial mistargeting of AGXT*LTM is observed, because the protein shows a localization different from the Mitotracker labeling and similar to the pattern obtained with wild-type AGXT. (Scale bar units are micrometers.)

Fig. 2.

(A) SDS/PAGE and fluorography of in vitro-synthesized AGXT proteins subject to 12 μg/ml trypsin for 10 min show significantly higher proteolytic sensitivity for variants carrying the P11L polymorphism, which is potentiated by the coexistence of the I244T mutation. (B) Immunoprecipitation of in vitro-synthesized wild-type AGXT and AGXT*LTM after 2–6 h of incubation at 30°C and reaction with either anti-Hsc70 or anti-Hsp90 antibodies; the mutant allele shows a much more stable association with both molecular chaperones than the wild type, and the association with Hsp90 can be inhibited specifically with 11 μg/ml geldanamycin. (C) The lane TnT represents half the input used for each immunoprecipitation. (D) FRA of nondenaturing lysates from COS7 cells transfected with plasmids containing either the wild-type AGXT or AGXT*LTM cDNA. Duplicate filters probed with either anti-Flag or anti-Hsp90 antibodies show that cells transfected with AGXT*LTM cDNA contain both AGXT and Hsp90 in aggregates retained in the 0.2-μm-pore membrane.

Fig. 3.

(A) Western blot of COS7 cells transfected with plasmids containing either the wild-type AGXT cDNA or cDNAs carrying the P11L polymorphism alone (*L), the I244T mutation alone (*T), or the haplotype found in our patients (*LTM); anti-Flag immunostaining was used to detect the AGXT variants and anti-actin antibody was used as a control in a replicate gel run in parallel. (B) Anti-Flag immunoprecipitation of 50 μg of total protein from 35S-Met-labeled cells expressing AGXT, *T, or *LTM after chase times of 30 min, 24 h, and 48 h; 1/20th of the labeled lysate used for immunoprecipitation was run in parallel as a control. (C) 35S-autoradiogram of total lysate. No significant differences are observed in the stability of the protein containing the I244T mutation alone or in combination with the P11L and I340M polymorphisms, relative to the wild-type protein.

Fig. 4.

(A) Western blot of COS7 cells transfected with Flag-tagged plasmids containing either the wild-type AGXT or AGXT*LTM cDNA. Two days after transfection, three aliquots of the cells were extracted with lysis buffers without detergents (sol.) or containing either 1% SDS or 6 M urea + 0.5% SDS (urea); mouse anti-Flag immunostaining was used to detect the AGXT variants, whereas rabbit anti-Ku70 antibody was used as a control and a replicate gel was stained with Coomassie blue. COS indicates untransfected cells. When followed over time, the differences in solubility of the expressed AGXT*LTM protein in sol. or containing 0.5% SDS become more evident 48 and 72 h after transfection (B), whereas no differences are found with the wild-type construct (C).

Fig. 5.

(A) Effect of various chemical chaperones on AGXT*LTM solubility. COS7 cells were transfected with Flag-tagged AGXT*LTM cDNA, divided into aliquots, and grown for 3 days in medium with 75 mM betaine, 80 μM pyridoxal phosphate (PyrP), 2.5 mM aminooxyacetic acid (AOA), 5% glycerol (Glyc.), 100 mM DMSO, 75 mM trimethylamine oxide (TMAO), or 10 mM phenylbutyric acid (PBA). Cell lysates were prepared with buffer without detergent, and 20 μg of protein was analyzed by Western blotting with anti-Flag antibody. The left lanes contain lysates of nontransfected COS7 cells or cells transfected with either wild-type AGXT or AGXT*LTM cDNA and grown in medium without additives, respectively. Anti-Ku70 antibody was used as a control. (B) Effect of temperature on AGXT*LTM solubility. COS7 cells were transfected with Flag-tagged plasmids containing either the wild-type AGXT or AGXT*LTM cDNA, divided into aliquots and incubated at either 37°C or 30°C. Four days after transfection, aliquots of the cells were extracted with lysis buffers containing either no detergent (soluble) or 0.5% SDS. Anti-Ku70 antibody was used as a control. Native lysates of cells expressing either AGXT or AGXT*LTM also were analyzed in parallel by Western blotting (C) and FRA (D) to ascertain the effect of betaine treatment on the solubility of the expressed protein. Betaine treatment consistently increased the AGXT*LTM Western blot signal detected in cell lysates without detergent. The same lysates (Sol.) showed, by FRA, a parallel reduction in the amount of insoluble aggregates after betaine treatment. This reduction of retained AGXT*LTM aggregates also is detected in cell extracts containing 1% SDS.

Fig. 6.

Interaction between wild-type AGXT and the mutant AGXT*LTM proteins. (A) Coimmunoprecipitation of proteins expressed in COS7 cells grown in the presence of 75 mM betaine to enhance the proportion of soluble AGXT*LTM protein. Transfection was performed with combinations of AGXT and AGXT*LTM cDNAs tagged either at the N terminus with Flag epitope or at the C terminus with V5 epitope, as indicated at the top. Two days after transfection, the cells were lysed in 0.5% Triton X-100, and proteins were immunoprecipitated with anti-Flag agarose beads. The autoradiogram represents a Western blot analysis with anti-V5 antibody of untransfected cells (COS) and the various lysates (before immunoprecipitation), wash buffer (wash), and immunoprecipitated protein (FlagIP). The presence of either tag does not seem to affect the predicted dimerization of wild-type AGXT, and similar dimerization can be observed with the mutant AGXT*LTM. Heterodimers between both protein variants also are observed. (B) Yeast two-hybrid interaction between various forms of the AGXT protein. Cotransfection of S. cerevisiae was performed with plasmids encoding Gal4 and lexA fusion proteins. Gal4 fusion constructs were produced with either wild-type AGXT or cDNAs carrying each of the amino acid changes (*L = P11L, *T = I244T, and *M = I340M) as well as a combination of all three (*LTM). lexA fusion constructs were produced with either wild-type AGXT or AGXT*LTM cDNAs. All protein interactions seem possible, as revealed by the positive X-gal staining of the transfectants, compared with negative controls (constructs with lexA cDNA and constructs with an unrelated human cDNA, RAD1).