Linda C Hsieh-Wilson

CiteSeerX

Update TRENDS in Biotechnology Vol.22 No.10 October 2004 4896 Jaber, M. et al. (1996) Essential role of b-adrenergic receptor kinase-1
in cardiac development and function. Proc. Natl. Acad. Sci. U. S. A. 93,
12974–12979
7 Rockman, H.A. et al. (1998) Control of myocardial contractile function
by the level of b-adrenergic receptor kinase-1 in gene-targeted mice.
J. Biol. Chem. 273, 18180–18184
8 Ungerer, M. et al. (1993) Altered expression of b-adrenergic receptor
kinase and b1-adrenergic receptors in the failing heart. Circulation
87, 454–463
9 Rockman, H.A. et al. (1998) Expression of a beta-adrenergic receptor
kinase 1 inhibitor prevents the development of myocardial failure in
gene-targeted mice. Proc. Natl. Acad. Sci. U. S. A. 95, 7000–7005
10 Harding, V.B. et al. (2001) Cardiac beta ARK1 inhibition prolongs
survival and augments beta blocker therapy in a mouse model of
severe heart failure. Proc. Natl. Acad. Sci. U. S. A. 98, 5809–5814Corresponding author: Linda C. Hsieh–Wilson (lhw@caltech.edu).
Available online 28 August 2004
www.sciencedirect.com11 Freeman, K. et al. (2001) Alterations in cardiac adrenergic signaling
and calcium cycling differentially affect the progression of cardiomyo-
pathy. J. Clin. Invest. 107, 967–974
12 Williams, M.L. and Koch, W.J. (2004) Viral-based myocardial gene
therapy approaches to alter cardiac function. Annu. Rev. Physiol. 66,
49–75
13 Iaccarino, G. et al. (1998) Reciprocal in vivo regulation of myocardial G
protein-coupled receptor kinase expression by b-adrenergic receptor
stimulation and blockade. Circulation 98, 1783–17890167-7799/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tibtech.2004.08.004Tailor-made glycoproteins
Linda C. Hsieh–Wilson
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USAPosttranslational modifications are a fundamental
mechanism for the regulation of cellular physiology
and function. A recent paper by Zhang et al. provides a
novel strategy for the generation of homogeneous
glycoproteins. The ability to install covalent modifi-
cations site-specifically into proteins holds tremendous
promise for deciphering the role of posttranslational
modifications and has exciting implications for the
development of protein therapeutics.
Given that the complexity of higher organisms is encoded
in a remarkably small number of genes, attention has
focused on other mechanisms to account for the diversity
of biological systems. Protein posttranslational modifi-
cations (PTMs) represent a major source of variation – the
covalent attachment of phosphate, lipid, and other
chemical groups to amino acid side chains extends the
capabilities of proteins and provides selective and tem-
poral control over protein function. At a cellular level,
PTMs have been shown to regulate vital processes such
as transcription, cell-cycle progression, and programmed
cell death.
Protein glycosylation, the most abundant and complex
form of PTM, requires an estimated 1% of mammalian
genes [1] and distinguishes eukaryotic cells from simpler
prokaryotic systems. Recent studies are challenging early
views of carbohydrates as non-specific, static structures
and are revealing exciting physiological roles for glyco-
sylation. For example, Wei et al. have shown that HIV
evades the immune system by evolving a dynamically
changing shield of carbohydrates [2]. Glycosamino-
glycans, another class of carbohydrates, play crucial
roles during development, with genetic disruption ofglycosaminoglycan biosynthesis associated with severe
growth defects [3]. The complex sulfation patterns present
in glycosaminoglycans, which are tissue-specific, tightly
regulated, and crucial for growth factor activation,
suggest an intricate relationship between carbohydrate
structure and biological function [3]. In the intracellular
context, dynamic forms of glycosylation have been identi-
fied, such as the addition of b-N-acetylglucosamine to
serine or threonine residues of proteins (O-GlcNAc
glycosylation) [4]. Recent studies have linked O-GlcNAc
glycosylation to the regulation of transcription factors
such as cAMP-responsive element binding protein (CREB)
[5] and the proteasome [6], raising the exciting possibility
that this carbohydrate modification plays a fundamental
role in cellular communication and protein function.
Despite many important contributions, the diverse
roles of glycosylation and other covalent modifications
are only beginning to be understood. Detailed studies of
their biological effects have been hindered by the dynamic
nature and complexity of PTMs in vivo. For instance, the
presence of a given modification can depend on the cell
type or status (e.g. stimulated vs non-stimulated, normal
vs diseased). It can also be restricted to subcellular
compartments or protein subpopulations and can occur
in combination with other PTMs. Except for protein
phosphorylation, in which an aspartic or glutamic acid
can sometimes mimic the phosphorylated residue, no
natural amino acid can serve as an effective surrogate
for glycosylation and other PTMs. Moreover, efforts to
purify modified proteins for biochemical studies gener-
ally require abundant sources and extensive purification.
Even when available, the purified proteins are fre-
quently heterogeneous, bearing incomplete or multiple
modifications.
A recent paper by Schultz and colleagues provides a
novel solution to these problems and should considerably
Update TRENDS in Biotechnology Vol.22 No.10 October 2004490enhance our ability to analyze and manipulate glyco-
protein structure and function [7]. Zhang et al. hijacked
the translational machinery of Escherichia coli to selec-
tively incorporate a glycosylated amino acid (serine
modified by b-GlcNAc, Figure 1) into the protein myo-
globin. The strategy produced a homogeneous, structur-
ally defined glycoprotein with the carbohydrate installed
at a pre-selected site.Hijacking the translational machinery
The key to their approach was the evolution of an
orthogonal synthetase-tRNA pair that genetically encodes
a glycosylated amino acid in response to the amber codon
TAG (Figure 1). Synthetases usually charge tRNA
molecules with one of the twenty naturally occurring
amino acids. To coax a synthetase into accepting an
unnatural glycosylated amino acid, residues within the
tyrosine-binding site of Methanococcus jannaschii tyrosyl
tRNA synthetase (TyrRS) were randomized to create a
large library of mutants. This library was then subjected to
a positive selection to enrich TyrRS mutants competent to
transfer the glycosylated amino acid to the M. jannaschii
suppressor tRNA, followed by a negative selection to
delete clones that transfer endogenous amino acids. After
several rounds of selection, a TyrRS mutant was identified
that selectively incorporated b-GlcNAc-serine into myo-
globin. With the evolved TyrRS synthetase, milligram
quantities of homogeneous glycoprotein could be gener-
ated by co-expression of the synthetase, suppressor tRNA,
and TAG-mutated myoglobin genes in E. coli in medium
containing the glycosylated amino acid. Notably, several
laboratories have demonstrated the power of hijacking the
translational machinery to provide insights into protein
structure and function [8–12]. By extending the strategy
now to glycoproteins, Zhang et al. illustrate the breadth of
the approach and its potential to transform our under-
standing of PTMs.TRENDS in Biotechnology 
OHO
HO
OH
O O
OH
1
O
O O
OH
2
Unnatural
amino acid
Orthogonal
synthetase
ATP
Suppressor
tRNA
CUA
anti-codon
TAG
codon
Protein with unnatural
glycosylated amino acid
incorporated at desired site
AcHN
AcHN
NH2
NH2
OAc
AcO
AcO
mRNA
Figure 1. Strategy for the selective incorporation of glycosylated amino acids into
proteins. An orthogonal tRNA synthetase capable of charging a suppressor tRNA
with the glycosylated amino acid 1 was selected from a library of mutants.
Milligram quantities of homogeneous glycoprotein were obtained upon co-
expression of the synthetase, suppressor tRNA, and TAG-mutated myogloblin
genes in E.coli in the presence of acetylated amino acid 2. Acetylation enhanced
transport of the glycosylated amino acid across cell membranes.
www.sciencedirect.comThe promise of homogeneous, modified proteins
The approach developed by Schultz and co-workers has
the advantages of high selectivity, efficiency, and yield. No
other glycoforms or unmodified forms of myoglobin were
generated. Because the overproduction of proteins in
E. coli is well established, the approach can also be opti-
mized to permit large-scale production of desired glyco-
proteins. In principle, the same synthetase could be used
to incorporate a glycosylated amino acid into other
proteins of interest or into multiple sites within the
same protein. These glycosylated amino acids could then
be further elaborated to more complex carbohydrate
structures using glycosyltransferase enzymes, as illus-
trated by a recent study from the Schultz and Wong
groups [13]. In the future, the selection strategy could
be applied to evolve tRNA synthetases for other PTMs
such as phosphorylation, acetylation, and methylation.
Finally, extension of the approach to other codon strate-
gies [11,12,14] could permit the introduction of multiple,
different PTMs into the same protein.
The strategy reported by Zhang et al. represents one of
several methods under active investigation for the
preparation of homogeneously modified proteins. Peptides
and small proteins are accessible via chemical synthesis,
but issues of cost and homogeneity complicate applica-
tion to larger proteins or commercial production. Chemical
methods have also been developed for the introduction
of PTM mimics via site-directed mutagenesis and
chemoselective labeling reagents [15,16]. Semi-synthetic
approaches, such as expressed protein ligation, have
permitted modified synthetic peptides containing phos-
phorylated, glycosylated or lipidated amino acids to be
coupled to recombinant proteins [17]. In a recent impres-
sive achievement, Kochendoerfer et al. used native
chemical ligation to construct the 166-amino acid erythro-
poietin protein with two monodisperse polymers attached
at natural glycosylation sites [18]. The resulting protein
displayed potent hematopoietic activity and an extended
time action in vivo.
The ability to generate homogeneous proteins bearing
specific modifications has tremendous potential for
advancing our understanding of PTMs. The incorporation
of covalent modifications at specific sites circumvents the
challenges of heterogeneous biochemical preparations.
The strategy should also permit studies of the interplay
among different modifications, such as the influence of one
modification on the location or kinetics of another. For
instance, phosphorylation and O-GlcNAc glycosylation
can occur at distinct sites within the protein, yet
functionally oppose one another [4,5]. In such cases, it
will be interesting to examine whether glycosylation
antagonizes the kinetics of phosphorylation and vice
versa. With the development of methods for incorporating
multiple PTMs into proteins, the combinatorial effects of
distinct modifications within a given protein can also be
addressed [19]. Such methods would be useful, for
example, to deconvolute the influence of histone acetyl-
ation, methylation, and phosphorylation on transcription.
Finally, recent extension of the unnatural amino acid
technology to yeast and mammalian cells [20–22] could
permit the expression of modified, activated forms of
Update TRENDS in Biotechnology Vol.22 No.10 October 2004 491proteins or dominant-negative mutants to understand
PTMs in a cellular context.
Methods for generating homogenously modified pro-
teins also have implications for protein therapeutics.
Nearly all therapeutic glycoproteins exist as hetero-
geneous mixtures of glycoforms, which can have profound
effects on the stability, solubility, and pharmacological
properties of the protein [23]. The ability to install
carbohydrates at specific sites could transform the way
in which therapeutic glycoproteins are discovered, devel-
oped, and manufactured. In this regard, parallels can be
drawn to small molecule pharmaceuticals, in which the
ability to evaluate single enantiomers established new
standards for the industry. If similar stringencies are
applied to protein therapeutics, the development of
methods for the production of homogeneous proteins will
become a major focus. Finally, the potential to fine-tune
the properties of proteins (e.g. potency, stability, time
action), much in the same way the medicinal chemist
improves the performance of small molecule drugs, could
lead to significant breakthroughs. By providing a new
method for producing homogeneous glycoproteins, the
work of Zhang et al. represents an important milestone
along this path.References
1 Lowe, J.B. and Marth, J.D. (2003) A genetic approach to mammalian
glycan function. Annu. Rev. Biochem. 72, 643–691
2 Wei, X.P. et al. (2003) Antibody neutralization and escape by HIV-1.
Nature 422, 307–312
3 Selleck, S.B. (2000) Proteoglycans and pattern formation - sugar
biochemistry meets developmental genetics. Trends Genet. 16,
206–212
4 Slawson, C. and Hart, G.W. (2003) Dynamic interplay between
O-GlcNAc and O-phosphate: the sweet side of protein regulation.
Curr. Opin. Struct. Biol. 13, 631–636
5 Lamarre-Vincent, N. and Hsieh-Wilson, L.C. (2003) Dynamic glyco-
sylation of the transcription factor CREB: A potential role in gene
regulation. J. Am. Chem. Soc. 125, 6612–6613Corresponding author: Melinda M. Pettigrew (melinda.pettigrew@yale.edu).
Available online 28 August 2004
www.sciencedirect.com6 Zhang, F.X. et al. (2003) O-GIcNAc modification is an endogenous
inhibitor of the proteasome. Cell 115, 715–725
7 Zhang, Z.W. et al. (2004) A new strategy for the synthesis of
glycoproteins. Science 303, 371–373
8 Beene, D.L. et al. (2003) Unnatural amino acid mutagenesis in
mapping ion channel function. Curr. Opin. Neurobiol. 13, 264–270
9 Mendel, D. et al. (1995) Site-directed mutagenesis with an expanded
genetic code. Annu. Rev. Biophys. Biomol. Struct. 24, 435–462
10 Link, A.J. et al. (2003) Non-canonical amino acids in protein
engineering. Curr. Opin. Biotechnol. 14, 603–609
11 Hohsaka, T. and Sisido, M. (2002) Incorporation of non-natural amino
acids into proteins. Curr. Opin. Chem. Biol. 6, 809–815
12 Anderson, R.D. et al. (2002) Fluorescence resonance energy transfer
between unnatural amino acids in a structurally modified dihydro-
folate reductase. J. Am. Chem. Soc. 124, 9674–9675
13 Liu, H.T. et al. (2003) A method for the generation of glycoprotein
mimetics. J. Am. Chem. Soc. 125, 1702–1703
14 Anderson, J.C. et al. (2004) An expanded genetic code with a
functional quadruplet condon. Proc. Natl. Acad. Sci. U. S. A. 101,
7566–7571
15 Davis, B.G. (2003) Chemical modification of biocatalysts. Curr. Opin.
Biotechnol. 14, 379–386
16 Hang, H.C. and Bertozzi, C.R. (2001) Chemoselective approaches to
glycoprotein assembly. Acc. Chem. Res. 34, 727–736
17 Muir, T.W. (2003) Semisynthesis of proteins by expressed protein
ligation. Annu. Rev. Biochem. 72, 249–289
18 Kochendoerfer, G.G. et al. (2003) Design and chemical synthesis of a
homogeneous polymer-modified erythropoiesis protein. Science 299,
884–887
19 Khidekel, N. and Hsieh-Wilson, L.C. (2004) A ‘molecular switchboard’
- covalent modifications to proteins and their impact on transcription.
Org. Biomol. Chem. 2, 1–7
20 Chin, J.W. et al. (2003) An expanded eukaryotic genetic code. Science
301, 964–967
21 Monahan, S.L. et al. (2003) Site-specific incorporation of unnatural
amino acids into receptors expressed in mammalian cells. Chem. Biol.
10, 573–580
22 Kohrer, C. et al. (2003) A possible approach to two different unnatural
site-specific insertion of amino acids into proteins in mammalian cells
via nonsense suppression. Chem. Biol. 10, 1095–1102
23 Haselbeck, A. (2003) Epoetins: differences and their relevance to
immunogenicity. Curr. Med. Res. Opin. 19, 430–432
0167-7799/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tibtech.2004.08.009An array of diverse microbial genomes
Melinda M. Pettigrew
Department of Epidemiology and Public Health, Yale University School of Medicine, New Haven, CT, 06520-8034, USAMethods for comparing gene frequencies across large,
epidemiologically defined bacterial collections are
limited. A novel microarray technology has been devel-
oped called ‘library on a slide’. In this technology,
hundreds of entire microbial genomes are arrayed,
rather than sequences of a single genome or sets of
genes. These slides can then be probed for the presence
of specific genes allowing researchers to drawinferences regarding important differences between
related strains that differ in their pathogenic potential.
Within a given bacterial species, individual strains often
exhibit incredible levels of diversity and can differ in genome
make-up by up to 20% [1]. Genome sizes can vary by as much
as 1 Mb among E. coli isolates, translating into a difference
of 800 genes between the largest and smallest strains [2].
Zhang et al. [3] at the University of Michigan have designed
a novel DNA microarray technology for exploring genetic
diversity among hundreds of closely related bacterial


Tailor-made glycoproteins

http://citeseerx.ist.psu.edu/viewdoc/download;jsessionid=94B1D0936421686314B73444DB6C95D1?doi=10.1.1.1042.1664&rep=rep1&type=pdf

Tailor-made glycoproteins

Abstract

Similar works

Full text

Available Versions

CiteSeerX