Impact of natural mutations on the riboflavin transporter 2 and their relevance to human riboflavin transporter deficiency 2

Abstract

Riboflavin transporter deficiency 2 (RTD2) is a rare neurological disorder caused by mutations in the Solute carrier family 52 member 2 (Slc52a2) gene encoding human riboflavin transporter 2 (RFVT2). This transporter is ubiquitously expressed and mediates tissue distribution of riboflavin, a water-soluble vitamin that, after conversion into FMN and FAD, plays pivotal roles in carbohydrate, protein, and lipid metabolism. The 3D structure of RFVT2 has been constructed by homology modeling using three different templates that are equilibrative nucleoside transporter 1 (ENT1), Fucose: proton symporter, and glucose transporter type 5 (GLUT5). The structure has been validated by several approaches. All known point mutations of RFVT2, associated with RTD2, have been localized in the protein 3D model. Six of these mutations have been introduced in the recombinant protein for functional characterization. The mutants W31S, S52F, S128L, L312P, C325G, and M423V have been expressed in E. coli, purified, and reconstituted into proteoliposomes for transport assay.All the mutants showed impairment of function. The K m for riboflavin of the mutants increased from about 3 to 9 times with respect to that of WT, whereas V max was only marginally affected. This agrees with the improved outcome of most RTD2 patients after administration of high doses of riboflavin.

Strategies for Successful Over-Expression of Human Membrane Transport Systems Using Bacterial Hosts: Future Perspectives

“…The protein was purified by nickel-chelating affinity chromatography and reconstituted in proteoliposomes in a folded state; this allowed the discovery of novel aspects of the transport mechanism, such as inhibition by FMN and the interaction with Ca 2+ ions [ 60 ]. Moreover, structure/function relationships were characterized thanks to the bacterially expressed proteins [ 59 , 60 ].…”

In silico investigation on structure–function relationship of members belonging to the human SLC52 transporter family

“…This disease can lead to blindness, deafness, and severe nerve palsies, but it can be in some cases successfully treated by high doses of vitamin B2 supplements. The pathogenic mutations in SLC52A2 and SLC52A3 described to date are reported in Console et al, 15 Tolomeo et al, 8 CureRTD database 16…”

“…The goal of this investigation is to obtain more reliable 3D models of RFVT1, RFVT2 and RFVT3, using in silico molecular modeling methods 2,10,15,21–27 . We propose here to improve the reliability of structural models by using innovative ab initio methods based on artificial intelligence (AI), including AlphaFold (AF) 28 and the newly released RoseTTaFold 29 .…”

Combined isobutyryl‐CoA and multiple acyl‐CoA dehydrogenase deficiency in a boy with altered riboflavin homeostasis

“…Recently, mutations in genes involved in Rf transport and metabolism, that is, Rf transporter 1 gene ( SLC52A1 ), 1 , 8 , 9 FAD synthase gene ( FLAD1 ), 10 and mitochondrial FAD transporter gene ( SLC25A32 ) 11 , 12 have been shown to cause MADD(‐like) clinical and biochemical phenotypes. Mutations in Rf transporter 3 gene ( SLC52A3 ) 13 and Rf transporter 2 gene ( SLC52A2 ), 14 which have been shown to cause Brown–Vialetto–Van Laere or Fazio Londe syndromes, might lead to a MADD(‐like) biochemical profile (see Tolomeo and colleagues 15 , 16 , 17 ). This is essential because FAD, synthesized from Rf, is the main cofactor for the functioning of ETF, ETF:QO and all ACADs.…”