Abstract
Antibodies to the Vel blood group antigen can cause adverse hemolytic reactions unless Vel-negative blood units are transfused. Since the genetic background of Vel-negativity was discovered in 2013, DNA-based typing of the 17-bp deletion causing the phenotype has facilitated identification of Vel-negative blood donors. SMIM1, the gene underlying Vel, encodes a 78-amino acid erythroid transmembrane protein of unknown function. The transmembrane orientation of SMIM1 has been debated since experimental data supported both the N- and C-termini being extracellular. Likewise, computational predictions of its orientation were divided and potential alternatives such as monotopic or dual-topology have been discussed but not investigated.We used a cell-free system to explore the topology of SMIM1 when synthesized in the endoplasmic reticulum (ER). SMIM1 was tagged with an opsin-derived N-glycosylation reporter at either the N- or C-terminus and synthesized in vitro using rabbit reticulocyte lysate supplemented with canine pancreatic microsomes as a source of ER membrane. SMIM1 topology was then determined by assessing the N-glycosylation of its N- or C-terminal tags. Complementary experiments were carried out by expressing the same SMIM1 variants in HEK293T/17 cells and establishing their membrane orientation by immunoblotting and flow cytometry.Our data consistently indicate that SMIM1 has its short C-terminus located extracellularly and that it most likely belongs to the tail-anchored class of membrane proteins with the bulk of the polypeptide located in the cytoplasm. Having established its membrane orientation in an independent model system, future work can now focus on functional aspects of SMIM1 as a potential regulator of erythropoiesis.
Highlights
Antibodies directed toward the Vel blood group antigen were first described in 1952 as a result of hemolytic transfusion reactions [1]
The Small Integral Membrane Protein 1 (SMIM1) gene, previously known by the name LOC388588 was thereby annotated in the human genome, which opened up the way for the genetic correlation between the 17-bp deletion in SMIM1 and the Vel– phenotype
The in vitro expression of SMIM1 with opsin tags to allow for N-glycosylation showed a clear preference for SMIM1 to orient in cellular membranes with its N-terminus located in the cytosol, resulting in its C-terminus being extracellular following the trafficking of newly synthesized SMIM1 to the plasma-membrane
Summary
Antibodies directed toward the Vel blood group antigen were first described in 1952 as a result of hemolytic transfusion reactions [1]. The SMIM1 gene consists of four exons and encodes a 78-amino acid long protein with a transmembrane region residing close to the C-terminus. The topology of the membrane inserted protein has been enigmatic, with multiple computational algorithms predicting different results. Depending on which algorithm was used, different orientations of the SMIM1 protein were predicted upon discovery of the molecule [7]. Another approach to suggest membrane orientation is the calculated free energy change associated with the entry of specific amino acid sequences into the lipid bilayer via the Sec translocon (Figure 1A) [8]
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