The ins and outs of SMIM1 and its relationship to the expression of Vel blood group antigen
Nylander, Anja LU
; Storry, Jill
LU
and Olsson, Martin L
LU
(2017)
27th Regional Congress of the International Society of Blood Transfusion, ISBT 2017
In Vox Sanguinis
112(Suppl. 1).
p.48-49
- Abstract
- Background: Vel blood group expression is dependent on Small Integral Membrane Protein 1 (SMIM1), a recently discovered erythroid protein. SMIM1 consists of 78 amino acids (aa) and shows only limited homology to other human proteins but is evolutionarily conserved, indicating its importance. The protein has a predicted transmembrane domain but the direction of insertion into the red blood cell (RBC) membrane is not yet clarified. The aa sequence contains reactive residues such as cysteines with the potential for disulphide bridge formation and a GXXXG motif known to induce dimerization in glycophorins. Many proteins form multimers to become functional. Thus, due to SMIM1's capability to dimerize and its association with mean corpuscular... (More)
- Background: Vel blood group expression is dependent on Small Integral Membrane Protein 1 (SMIM1), a recently discovered erythroid protein. SMIM1 consists of 78 amino acids (aa) and shows only limited homology to other human proteins but is evolutionarily conserved, indicating its importance. The protein has a predicted transmembrane domain but the direction of insertion into the red blood cell (RBC) membrane is not yet clarified. The aa sequence contains reactive residues such as cysteines with the potential for disulphide bridge formation and a GXXXG motif known to induce dimerization in glycophorins. Many proteins form multimers to become functional. Thus, due to SMIM1's capability to dimerize and its association with mean corpuscular haemoglobin concentration (MCHC), we hypothesized that the above-mentioned motifs are of importance. Aims: The aim of this study was to investigate two features of SMIM1; GXXXG and the cysteine residues. Through mutagenesis studies, we investigated their role in Vel antigen expression and dimerization. Methods: Glycine in the GXXXG motif (aa 67-71) of the predicted transmembrane portion was substituted for leucine. Cysteine at positions 35, 43 and 77 was substituted for alanine. The SMIM1 constructs (incl. mock and wild-type) were transiently expressed in K562 cells. Vel surface expression was analysed using flow cytometry (FACS) with human polyclonal anti-Vel. The total protein content and dimerization was analysed by Western blot with a rabbit polyclonal antibody produced against the N-terminus (aa 1-15) of SMIM1. α-chymotrypsin treatment was performed as described [Storry et al. Nat Genet, 2013]. Results: Mutation of either or both of the glycines in the GXXXG motif caused significant reduction of Vel surface expression compared to SMIM1 wild-type, although the SMIM1 protein content remained unchanged as evaluated by Western blot. Substitution of p.Cys77Ala also significantly reduced Vel antigen expression, while no effect was observed by p.Cys35Ala or p.Cys43Ala. Again, the SMIM1 content appeared unaffected by the substitutions. The capacity to form dimers remained intact in all mutants. To verify the location of the N-terminus, α-chymotrypsin treatment of intact RBCs was performed. Absence of SMIM1-characteristic bands on Western blot of RBC membranes indicated that the N-terminal target for the rabbit antibody used was destroyed. Summary / Conclusions: Vel surface expression is severely affected by substitutions of glycine at positions 67 and 71, or of cysteine at position 77 in SMIM1. Disruption of a transmembrane localisation signal may explain the importance of glycine substitutions. The importance of Cys77 for Vel antigenicity could indicate an extracellular location of the C-terminus. This appears to be consistent with conclusions from the study by Arnaud et al. [FEBS Lett., 2015]. However, this is somewhat contradicted by our results from α-chymotrypsin treatment of intact RBCs. If the C-terminus is indeed extracellular and SMIM1 a type II protein, the N-terminus should be insensitive to such treatment. In summary, we conclude that the targeted GXXXG motif and Cys77 are important for the correct, extracellular exposure of the Vel antigen. It remains unclear if SMIM1 is a type I or II transmembrane protein. This question deserves further investigation to facilitate studies on functional aspects of this enigmatic protein. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/291c86b1-d0b6-4131-9a81-859b3fb69671
- author
- Nylander, Anja
LU
; Storry, Jill
LU
and Olsson, Martin L
LU
- organization
- publishing date
- 2017
- type
- Contribution to journal
- publication status
- published
- subject
- in
- Vox Sanguinis
- volume
- 112
- issue
- Suppl. 1
- article number
- 4A-S20-01
- pages
- 48 - 49
- publisher
- Wiley-Blackwell
- conference name
- 27th Regional Congress of the International Society of Blood Transfusion, ISBT 2017
- conference location
- Copenhagen, Denmark
- conference dates
- 2017-06-17 - 2017-06-21
- external identifiers
-
- pmid:28608429
- ISSN
- 1423-0410
- DOI
- 10.1111/vox.12530
- language
- English
- LU publication?
- yes
- id
- 291c86b1-d0b6-4131-9a81-859b3fb69671
- date added to LUP
- 2017-12-19 16:33:08
- date last changed
- 2021-09-30 04:09:28
@misc{291c86b1-d0b6-4131-9a81-859b3fb69671,
abstract = {{Background: Vel blood group expression is dependent on Small Integral Membrane Protein 1 (SMIM1), a recently discovered erythroid protein. SMIM1 consists of 78 amino acids (aa) and shows only limited homology to other human proteins but is evolutionarily conserved, indicating its importance. The protein has a predicted transmembrane domain but the direction of insertion into the red blood cell (RBC) membrane is not yet clarified. The aa sequence contains reactive residues such as cysteines with the potential for disulphide bridge formation and a GXXXG motif known to induce dimerization in glycophorins. Many proteins form multimers to become functional. Thus, due to SMIM1's capability to dimerize and its association with mean corpuscular haemoglobin concentration (MCHC), we hypothesized that the above-mentioned motifs are of importance. Aims: The aim of this study was to investigate two features of SMIM1; GXXXG and the cysteine residues. Through mutagenesis studies, we investigated their role in Vel antigen expression and dimerization. Methods: Glycine in the GXXXG motif (aa 67-71) of the predicted transmembrane portion was substituted for leucine. Cysteine at positions 35, 43 and 77 was substituted for alanine. The SMIM1 constructs (incl. mock and wild-type) were transiently expressed in K562 cells. Vel surface expression was analysed using flow cytometry (FACS) with human polyclonal anti-Vel. The total protein content and dimerization was analysed by Western blot with a rabbit polyclonal antibody produced against the N-terminus (aa 1-15) of SMIM1. α-chymotrypsin treatment was performed as described [Storry et al. Nat Genet, 2013]. Results: Mutation of either or both of the glycines in the GXXXG motif caused significant reduction of Vel surface expression compared to SMIM1 wild-type, although the SMIM1 protein content remained unchanged as evaluated by Western blot. Substitution of p.Cys77Ala also significantly reduced Vel antigen expression, while no effect was observed by p.Cys35Ala or p.Cys43Ala. Again, the SMIM1 content appeared unaffected by the substitutions. The capacity to form dimers remained intact in all mutants. To verify the location of the N-terminus, α-chymotrypsin treatment of intact RBCs was performed. Absence of SMIM1-characteristic bands on Western blot of RBC membranes indicated that the N-terminal target for the rabbit antibody used was destroyed. Summary / Conclusions: Vel surface expression is severely affected by substitutions of glycine at positions 67 and 71, or of cysteine at position 77 in SMIM1. Disruption of a transmembrane localisation signal may explain the importance of glycine substitutions. The importance of Cys77 for Vel antigenicity could indicate an extracellular location of the C-terminus. This appears to be consistent with conclusions from the study by Arnaud et al. [FEBS Lett., 2015]. However, this is somewhat contradicted by our results from α-chymotrypsin treatment of intact RBCs. If the C-terminus is indeed extracellular and SMIM1 a type II protein, the N-terminus should be insensitive to such treatment. In summary, we conclude that the targeted GXXXG motif and Cys77 are important for the correct, extracellular exposure of the Vel antigen. It remains unclear if SMIM1 is a type I or II transmembrane protein. This question deserves further investigation to facilitate studies on functional aspects of this enigmatic protein.}},
author = {{Nylander, Anja and Storry, Jill and Olsson, Martin L}},
issn = {{1423-0410}},
language = {{eng}},
note = {{Conference Abstract}},
number = {{Suppl. 1}},
pages = {{48--49}},
publisher = {{Wiley-Blackwell}},
series = {{Vox Sanguinis}},
title = {{The ins and outs of SMIM1 and its relationship to the expression of Vel blood group antigen}},
url = {{http://dx.doi.org/10.1111/vox.12530}},
doi = {{10.1111/vox.12530}},
volume = {{112}},
year = {{2017}},
}