Analysis of the Qatari R336C cystathionine β‐synthase protein in mice

Abstract

Classical homocystinuria is a recessive inborn error of metabolism caused by mutations in the cystathionine beta‐synthase (CBS) gene. The highest incidence of CBS deficiency in the world is found in the country of Qatar due to the combination of high rates of consanguinity and the presence of a founder mutation, c.1006C>T (p.R336C). This mutation does not respond to pyridoxine and is considered severe. Here we describe the creation of a mouse that is null for the mouse Cbs gene and expresses human p.R336C CBS from a zinc‐inducible transgene (Tg‐R336C Cbs −/−). Zinc‐treated Tg‐R336C Cbs −/− mice have extreme elevation in both serum total homocysteine (tHcy) and liver tHcy compared with control transgenic mice. Both the steady‐state protein levels and CBS enzyme activity levels in liver lysates from Tg‐R336C Cbs −/− mice are significantly reduced compared to that found in Tg‐hCBS Cbs −/− mice expressing wild‐type human CBS. Treatment of Tg‐R336C Cbs −/− mice with the proteasome inhibitor bortezomib results in stabilization of liver CBS protein and an increase in activity to levels found in corresponding Tg‐hCBS Cbs −/− wild type mice. Surprisingly, serum tHcy did not fully correct even though liver enzyme activity was as high as control animals. This discrepancy is explained by in vitro enzymatic studies of mouse liver extracts showing that p.R336C causes reduced binding affinity for the substrate serine by almost 7‐fold and significantly increased dependence on pyridoxal phosphate in the reaction buffer. These studies demonstrate that the p.R336C alteration effects both protein stability and substrate/cofactor binding.

Examination of two different proteasome inhibitors in reactivating mutant human cystathionine β-synthase in mice

“…We next examined the ability of carfilzomib to rescue four additional mouse models of human CBS deficiency: Tg-R266K , Tg-I278T , Tg-R336C , and Tg-T191M . All but the Tg-T191M - mice have been previously described [ 12 , 13 , 16 ]. The Tg-T191M mouse expresses a non-pyridoxine responsive mutant CBS protein found at high frequency in patients from in Spanish speaking countries [ 20 ] and the creation is described in the Methods section.…”

“…The Tg-S466L Cbs -/- , Tg-R266K Cbs -/- , Tg-R336C Cbs -/- and Tg-I278T Cbs -/- mouse models have been previously described [ 12 , 13 , 15 , 16 ]. For simplification the Cbs -/- genotype has been omitted throughout the text.…”

The Spectrum of Mutations of Homocystinuria in the MENA Region

“…Clinically, patients with classic homocystinuria exhibit numerous manifestations during infancy because homocysteine disrupts the development of many organ systems. These include Marfanoid habitus, pectus exavatum (curved-in sternum), pectus carinatum (protruding sternum), genu valgum, where knees are angled toward each other, and many other skeletal deformities [67,68]. In addition, ectopia lentis (dislocation of the lens) can lead to nearsightedness and blurred vision.…”

Recent therapeutic approaches to cystathionine beta‐synthase‐deficient homocystinuria

“…Genetic analyses showed a consistent association of specific CBS variants with full or extreme responsiveness (e.g., p.P49L, p.A114V and p.I278T) or non‐responsiveness to pyridoxine (e.g., p.R125Q, p.E176K, p.T191M, p.T262M and p.G307S). In contrast to this consistent correlation of genotype‐to‐pyridoxine responsiveness in HCU patients, the insight gained from using in vitro, bacterial or eukaryotic expression systems and animal models of HCU have yielded discrepant results (Chen et al, 2006; Gupta et al, 2017, 2019; Majtan, Pey, Ereno‐Orbea, et al, 2016). Due to the discrepancy between clinical experience and research data, pyridoxine responsiveness in HCU remains mechanistically unclear.…”

“…Similarly, Tg‐R266K Cbs −/− mice expressing hCBS p.R266K mutant and treated with bortezomib exhibited 25‐fold increase in mean liver CBS activity compared to the untreated cohort with their mean serum tHcy entirely normalized to 6.7 μM (Gupta et al, 2017). Likewise, the Tg‐R336C Cbs −/− model expressing hCBS p.R336C mutant showed strong rescue by bortezomib with 10‐fold increase of their mean liver CBS activities (Gupta et al, 2019). The one transgenic HCU mouse model that was not rescued by PI treatment was the Tg‐G307S Cbs −/− mice expressing hCBS p.G307S mutant, an allele primarily found in pyridoxine unresponsive HCU patients of Irish origin (Gupta et al, 2018).…”