Functional characterization of variants in human TPO gene using baculovirus expression system.

MARICEL FERNANDA MOLINA; EZEQUIELA ADROVER; SEBATIAN GONZALEZ; ADRIANA VICTORIA SABLJIC; ALDANA TRABUCCHI; SILVINA NOEMÍ VALDEZ; RODOLFO GONZALEZ LEBRERO; ALEXANDRA MARISA TARGOVNIK; CARINA M. RIVOLTA

Mar del Plata

Congreso; LXVIII Reunión Científica Anual de la Sociedad Argentina de Investigación Clínica (SAIC).; 2023

Sociedad Argentina de Investigación Clínica (SAIC).

The congenital hypothyroidism (CH) is the most common endocrinedisease in children with a prevalence of 1 in 2000-3000 live births.Variants in Thyroid peroxidase (TPO) are more often associated withpermanent CH by iodide organification defect and are commonly inheritedin an autosomal recessive manner. TPO is a 933 amino acidslong membrane-bound glycoprotein located at the apical membraneof the thyroid follicular cells that plays a key role in thyroid hormonesbiosynthesis. The TPO enzyme activity depends on both properfolding and membrane insertion, and an intact catalytic site. The aimof this study was to investigate the functional impact of TPO genemissense mutations previously identified in our laboratory (p.Ala-576Glu, p.Arg595Lys and p.Val748Met) in patients with permanentCH. The baculovirus expression vector system (BEVS) was used toexpress the WT and mutated proteins. First, TPO cDNA obtained byRT PCR and sequential cloning strategy in pGEMT previously in ourlaboratory was cloned into the pFastBacTMDUAL expression vector.Then, site-directed mutagenesis was performed, and recombinantvirus were constructed using the Bac-to-Bac® Baculovirus ExpressionSystems (Invitrogen). Recombinant proteins were expressedin Sf9 insect cells, purified, and characterized by SDS-PAGE andWestern blot. Enzyme activity was determined by monitoring theoxidation reaction of the substrate guaiacol spectrophotometrically.The values of apparent Km and Vmax were determined using theGauss–Newton algorithm. Two of three mutants (p.Arg595Lys andp.Val748Met) were absent in the enriched membrane fraction. Thefunctional evaluation of the third mutant (p.Ala576Glu) showed decreasedactivity with respect to WT, with a higher Km value and lowerreaction efficiency (Vmax). The identification and characterizationof TPO variants is undoubtedly a valuable approach to study theTPO structure/function relations and for the elaboration of a clinicaldiagnosis and genetic counseling.