Selenoprotein Synthesis and Reactivity - Biotechnological and Biomedical
Applications
E.S.J. Arnér
Division of Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet,
SE-171 77 Stockholm, Sweden
(Email: elias.arner@ki.se)
Introduction
Selenoproteins contain the chemically reactive
selenium-containing amino acid selenocysteine
(Sec), which is co-translationally incorporated at a
UGA (opal) codon via intricate and unique Secdedicated synthesis machineries. Using a genetic
tailoring methodology that enables directed Secinsertion at a pre-defined UGA codon,
recombinant selenoproteins can be produced in E.
coli (Arnér, 2002). This technique may be
employed for studies of selenoprotein features and
detailed analyses of native selenoprotein kinetics,
but may also be utilized for the development of
new biomedical applications based upon the use
of Sec reactivity (Johansson et al., 2005). This
presentation will be a short survey of examples
utilizing recombinant selenoproteins for in-depth
studies and for the development of novel
technologies within nuclear medicine.
selenenylsulfide bridge between Cys497 and
Sec498 (Figure 1).
Figure 1. The selenenylsulfide at the C-terminus
of TrxR1, formed between the selenium atom of
Sec (red) and the sulfur atom of Cys (yellow).
This figure is based upon the crystal structure
reported by Cheng et al. (2009).
Use of a Sel-tag for Nuclear Imaging
In-depth Studies of Selenoprotein Properties
Recombinant protein production is a powerful
methodology enabling in-depth studies of enzyme
kinetics or other protein properties. Selenoproteins
are, however, not easily synthesized by regular
means due to their intricate translation. With
mammalian thioredoxin reductases (TrxR),
however, these enzymes are particularly suitable
for genetic engineering with production of the
Sec-containing protein due to their penultimate Cterminal location of the Sec residue (Arnér et al.,
1999) although similar methodology can also be
utilized to incorporate Sec within the internal
parts of a protein, but with additional technical
complications and lower yield (Arnér, 2002; Jiang
et al., 2004). Using this previously described
method, we were recently able to determine the
crystal structure of Sec-containing rat TrxR1 in
both oxidized and reduced form (Cheng et al.,
2009). This confirmed the existence of our
previously proposed ß turn-like bend of a
We reasoned that the same Sec-containing redox
active C-terminal motif as found naturally in
mammalian TrxR1 could potentially be utilized as
a multifunctional fusion motif for nonselenoproteins, called a Sel-tag. This motif would
enable use of the unique chemical properties of
Sec for novel biotechnological applications, such
as phenylarsine oxide (PAO) Sepharose-based
single-step purification, site-specific labeleing
with fluorescent probes or 75Se, or radiolabeling
with radionuclides suitable for Positron Emission
Tomography (PET) imaging. Indeed, all of these
potential applications have proven possible and
could not be duplicated with Sec-to-Cys
substituted variants of the same motif (Johansson,
Chen et al., 2004; Cheng et al., 2006; Cheng et al.,
2006). This versatility of the Sel-tag is
schematically summarized in Figure 2.
Figure 2. A scheme summarizing the structure,
reactivity and diversity of a Sel-tag compared to a
dithiol variant of the same motif. Figure taken
from Cheng et al. (2006).
Recent (yet unpublished) progress utilizing
studies with 11C-labeled Sel-tagged protein
ligands have proven these to be highly useful for
nuclear imaging with PET. Results of these
studies include the imaging of apoptotic tissue or
angiogenesis in vivo using rodent models. Work
for the development of clinical applications based
upon this methodology is on-going.
Conclusions
Recent studies regarding the evolution of
selenoproteins
strongly
suggest
that
selenocysteine can support functions in
selenoproteins that can not easily, or not at all, be
maintained by cysteine orthologues (Castellano
2009; Castellano et al., 2009). Although it is not
yet completely certain, in mechanistic terms, what
these unique Sec-maintained functions would be,
it seems reasonable to suggest that unique
chemical reactivities of Sec compared to Cys
serve the evolutionary pressure to synthesize Seccontaining proteins, in spite of their costly and
complex translation machineries. In the work
presented here, claims are made that these unique
Sec-dependent properties can be utilized for novel
biomedical applications, such as PET imaging
with Sel-tagged protein ligands, and that the
recombinant production of selenoproteins in E
coli enables the development of such
technologies.
References
Arnér, E.S.J. 2002. Recombinant expression of
mammalian
selenocysteine-containing
thioredoxin
reductase
and
other
selenoproteins in Escherichia coli. Methods
Enzymol, 347: 226-235.
Arnér, E.S.J., Sarioglu, H., Lottspeich, F.,
Holmgren, A., and Böck, A. 1999. High-level
expression
in
Escherichia
coli
of
selenocysteine-containing rat thioredoxin
reductase utilizing gene fusions with
engineered bacterial-type SECIS elements
and co-expression with the selA, selB and
selC genes. Journal Molecular Biology, 292:
1003-1016.
Castellano, S. 2009. On the unique function of
selenocysteine - Insights from the evolution
of selenoproteins. Biochim Biophys Acta.
doi:10.1016/j.bbagen.2009.03.027.
Castellano, S., Andres, A. M., Bosch, E., Bayes,
M., Guigó, R. and Clarket, A.G. 2009. Low
exchangeability of selenocysteine, the 21st
amino acid, in vertebrate proteins. Molecular
Biology and Evolusion, 26(9): 2031-2040.
Cheng, Q., Johansson, L., Thorell, J.O., et al.
2006. Selenolthiol and dithiol C-terminal
tetrapeptide motifs for one-step purification
and labeling of recombinant proteins
produced in E. coli. ChemBioChem, 7: 19761981.
Cheng, Q., Sandalova, T., Lindqvist, Y., and
Arnér, ESJ. 2009. Crystal structure and
catalysis of the selenoprotein thioredoxin
reductase 1. Journal of Biological Chemistry,
284(6): 3998-4008.
Cheng, Q., Stone-Elander, S., and Arnér, E.S.
2006. Tagging recombinant proteins with a
Sel-tag for purification, labeling with
electrophilic compounds or radiolabeling
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glutathione S-transferase in Escherichia coli.
Biochemical and biophysical research
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Johansson, L., Chen, C., Thorell, J.O., et al. 2004.
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Johansson, L., Gafvelin, G., and Arnér, E.S. 2005.
Selenocysteine in proteins - properties and
biotechnological
use.
Biochimica
et
biophysica acta, 1726(1): 1-13.
Evidences of Selenium Deficiency in Brazil: from Soil to Human Nutrition
M.F. Moraes1,*, R.M. Welch2, M.R. Nutti3, J.L.V. Carvalho3, and E. Watanabe3
1
University of Sao Paulo, Luiz de Queiroz College of Agriculture, Av. Padua dias, 11, Piracicaba, Brazil
2
USDA-ARS, Robert W. Holley Center for Agriculture and Health, Cornell University, Ithaca, USA
3
National Research Center for Food Technology, EMPRAPA, Rio de Janeiro, Brazil
(*Corresponding email: moraesmf@yahoo.com.br)
Introduction
Cereal production has kept pace with the human
population growth rate. It is anticipated that world
demand for food will double in the period from
1990 - 2030, the increase being 3.5 times in
developing countries (Daily et al., 1998).
Malnutrition has increased, reaching almost half
of the world’ population, particularly among
pregnant women, infants, and children (Welch,
2008). This is partly due to soil micronutrient
deficiency. Deficiencies of iron (Fe), iodine (I),
selenium (Se) and zinc (Zn) are today the major
concern in relation to human health especially in
developing countries. According to the World
Health Organization more than 2 billion people
could be anemic as a consequence of Fe
deficiency (Allen et al., 2006). It has been
suggested that one fifth of the population is not
ingesting adequate amounts of Zn (Hotz and
Brown, 2004). Combs (2001) estimated that
between 0.5 and 1.0 billion people could be
deficient in Se.
Selenium in Soils
A world soil-plant study conducted by Sillanpää
and Jansson (1992) included Brazil among the
low soil Se countries, together with Finland.
There are few reports of analyses of Se in
Brazilian soils (Table 1); however the data has
shown that many soils are in the deficient range.
Table 1. Total Se concentration in Brazilian soils.
State or city Se (µg kg-1)
Reference
Paiva Neto and
Sao Paulo
0 - 800
Gargantini (1956)
Goias
1-8
Fichtner et al. (1990)
Sao Paulo
Anno (2001)
38 - 212†
Nova Odessa
130†
M.A. Zanetti‡
Sao Paulo
68 - 220
Faria (2009)
Deficient range 100 - 600 Lyons et al. (2003)
†Se concentration in µg dm-3; ‡Personal communication Marcus A. Zanetti (USP).
Selenium in Agricultural Food Products
No complete surveys of the occurrence of Se
deficiency in the Brazilian population are
available. Nevertheless, Ferreira et al. (2002)
reported that Se in plant foods was considered
low, possibly because the soils were low in Se
(Table 2). Lucci et al. (1984) determined the Se
concentration in grasses and animal feedstuff from
80 locals in the State of Sao Paulo. They found
low Se in grasses, with an average of 66 µg kg-1,
and also Se was low in both grain and silage of
maize, with 31 and 40 µg kg-1, respectively.
Table 2. Se in agricultural food products of Brazil.
Plant
Se (µg kg-1)
Reference
Lucci et al.
Pasture grass
66
(1984)
Pasture grass
67 - 123
Anno (2001)
Martens et al.
14
Dried beans S1†
(2004)
Martens et al.
Dried beans S2
1,710
(2004)
Martens et al.
Dried beans S3
240
(2004)
Chang
et al.
30-31,700¶
Brazil nut S1‡
(1995)
1,250Chang et al.
Brazil nut S2
512,000¶
(1995)
Lucci et al.
Maize grains
31
(1984)
Ferreira et al.
Food crops
< 50
(2002)
†
Dried beans samples from: S1 - State of Rio Grande do Sul;
S2 - State of Ceara; S3 - State of Para. ‡Brazil nuts samples
from: S1 - States of Acre and Rondonia; S2 - States of
Amazonas and Para. ¶Fresh weight
Some agricultural foods have high Se
concentrations, but this depends on the Se status
of the soils that the crops were grown (Table 2).
Unfortunately, data for the soil-Se levels in the
states along with Se concentrations in agricultural
products (e.g., Amazonas, Para and Ceara) is not
available.
Daily Selenium Intakes
Studies on the intake of Se by the people in some
Brazilian states have showed a low intake of Se in
Sao Paulo and Mato Grosso (Table 3). However,
in the Amapa and Amazonas states Se intake is
adequate (Maihara et al., 2004).
Table 3. Daily Se intake of Brazilian people¶.
State or city
Average (µg d-1)
Manaus
94.5
Mato Grosso
19
Santa Catarina
52†
Santa Catarina
139††
São Paulo
18
São Paulo city (Children)
26.3
Macapá city (Children)
107
Belém city (Children)
37.4
¶
Sources: Adapted by Gonzaga et al. (2007) and Maihara et al.
(2004). †Low social class. ††High social class.
Although there are conclusive evidences of Se
deficiencies in Brazil, it has not included in the
HarvestPlus biofortification program of Brazil.
More research on soil-Se levels and Se contents in
agricultural products from all Brazilian states is
needed. It is well known that the application of Se
containing fertilizers is efficacious at correcting
low Se levels in human diets (such as in Finland,
New Zealand and Australia).
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