Contents
Why are Glycoproteins Modified by Poly-N-Acetyllactosamine
Glycoconjugates? Pp.1-9
Dapeng Zhou
Mapping Protein:
Carbohydrate Interactions
Pp.11-20
Gerald F. Audette,
Louis T.J. Delbaere and Jim Xiang
Bacterial b-ketoacyl-Acyl Carrier Protein Synthases as
Targets for Antibacterial Agents Pp.21-29
Neuropeptide
Conversion to Bioactive Fragments - An Important Pathway in Neuromodulation Pp.31-44
M. Hallberg and F.
Nyberg
Hormonal Control of
the Neuropeptide Y System
Pp.45-57
Paolo Magni
Structural/Functional
Aspects of ES-62 – A Secreted Immunomodulatory Phosphorylcholine-Containing
Filarial Nematode Glycoprotein Pp.59-71
W. Harnett, M. M. Harnett and O. Byron
Rapid Translation
System (RTS): A Promising Alternative for Recombinant Protein Production Pp.73-80
J.-M. Betton
Abstracts
Back to top] Why are
Glycoproteins Modified by Poly-N-Acetyllactosamine Glycoconjugates?
Dapeng Zhou
Poly-N-acetyllactosamine
structures occur in mammalian glycoproteins in both N- and O-linked glycans.
They represent a backbone for additional modifications by fucosyltransferases,
sialyltransferases and sulfotransferases. These glycans have been suggested to
be involved in biospecific interactions with selectins and other glycan-binding
proteins. Moreover, the poly-Nacetyllactosamine chains in N-glycans have been
found to promote tumor progression and metastasis. Poly-N-acetyllactosamine
chains are synthesized by repeated alternating additions of Nacetylglucosamine
and galactose, catalyzed by b-1,3-N-acetylglucosaminyltransferases
(poly-N-acetyllactosamine synthase) and b-1,4-galactosyltransferases.
This review describes the poly-N-acetyllactosamine assembling machinery and
focuses on recent advances in the molecular cloning and characterization of
poly-N-acetyllactosamine synthase gene families. Recent progress in revealing
the biological functions of poly-N-acetyllactosamine structures by various
approaches in vitro and in vivo using different model systems has also been
summarized.
Back to top] Mapping Protein:
Carbohydrate Interactions
Gerald F. Audette,
Louis T.J. Delbaere and Jim Xiang
Many biologically important
interactions occur between proteins and carbohydrates. The examination of these
interactions at the atomic level is critical not only in understanding the
nature of these interactions and their biological role, but also in the design
of effective modulators of these interactions. While experimentally obtained
structural information is preferred, quite often this information is
unavailable. In order to address this, several methods have been developed to
probe the interactions between protein and carbohydrate in the absence of
structural data. These methods map the interactions between protein and
carbohydrate, and identify the groups involved, both at the carbohydrate and protein
level. Here, we review these developments, and examine the strengths,
weaknesses, and pitfalls of these methods.
Back to top] Bacterial b-ketoacyl-Acyl Carrier Protein Synthases as
Targets for Antibacterial Agents
Sanjay S. Khandekar,
Robert A. Daines, and John T. Lonsdale
As a result of increasing drug
resistance in pathogenic bacteria, there is a critical need for novel
broad-spectrum antibacterial agents. As fatty acid synthesis (FAS) in bacteria
is an essential process for cell survival, the enzymes involved in the FAS
pathway have emerged as promising targets for antimicrobial agents. Several
lines of evidence have indicated that bacterial condensing enzymes are central
to the initiation and elongation steps in bacterial fatty acid synthesis and
play a pivotal role in the regulation of the entire fatty acid synthesis
pathway. b-ketoacyl-acyl carrier
protein (ACP) synthases (KAS) from various bacterial species have been cloned,
expressed and purified in large quantities for detailed enzymological,
structural and screening studies. Availability of purified KAS from a variety
of bacteria, along with a combination of techniques, including combinatorial
chemistry, high-throughput screening, and rational drug design based on crystal
structures, will undoubtedly aid in the discovery and development of much
needed potent and broadspectrum antibacterial agents. In this review we
summarize the biochemical, biophysical and inhibition properties of b-ketoacyl-ACP synthases from a variety of
bacterial species.
Back to top] Neuropeptide
Conversion to Bioactive Fragments - An Important Pathway in Neuromodulation
M. Hallberg and F.
Nyberg
Biosynthetic pathways for the
formation of neuroactive peptides and the processes for their inactivation
include several enzymatic steps. In addition to enzymatic processing and
degradation, several neuropeptides have been shown to undergo enzymatic
conversion to fragments with retained or modified biological activity. This has
most clearly been demonstrated for e.g. opioid peptides, tachykinins,
calcitonin gene-related peptide (CGRP) as well as for peptides belonging to the
renin-angiotensin system. Sometimes the released fragment shares the activity
of the parent compound. However, in many cases the conversion reaction is
linked to a change in the receptor activation profile, i.e. the generated
fragment acts on and stimulates a receptor not recognized by the parent
peptide. This review will describe the characteristics of certain neuropeptide
fragments having the ability to modify the biological action of the peptide
from which they are derived. Focus will be directed to the tachykinins, the
opioid peptides, angiotensins as well as to CGRP, bradykinin and nociceptin.
The k opioid receptor selective opioid peptide, dynorphin, recognized for its
ability to produce dysphoria, is converted to the d opioid receptor agonist Leu-enkephalin, with euphoric
properties. The tachykinins, typified by substance P (SP), is converted to the
bioactive fragment SP(1-7), a heptapeptide mimicking some but opposing other
effects of the parent peptide. The bioactive angiotensin II, known to bind to
and stimulate the AT-1 and AT-2 receptors, is converted to angiotensin IV (i.e.
angiotensin 3-8) with preference for the AT-4 sites or to angiotensin (1-7),
not recognized by any of these receptors. Both angiotensin IV and angiotensin
(1-7) are biologically active. For example angiotensin (1-7) retains some of
the actions ascribed for angiotensin II but is shown to counteract others.
Thus, it is obvious that the activity of many neuroactive peptides is modulated
by bioactive fragments, which are formed by the action of a variety of
peptidases. This phenomenon appears to represent an important regulatory
mechanism that modulates many neuropeptide systems but is generally not
acknowledged.
Back to top] Hormonal Control
of the Neuropeptide Y System
Paolo Magni
Neuropeptide Y (NPY) and the related receptors represent a widely diffused system that is involved in the regulation of multiple biological functions. NPY, a 36-aminoacid peptide expressed in several areas of the nervous system, is a pleiotropic factor participating to the control of some physiological processes, such as cognitive functions, eating behavior, circadian rhythms, neuroendocrine mechanisms, reproductive and cardiovascular functions. NPY acts through a series of G-protein-associated membrane receptors (NPY-Rs), characterized by different tissue distribution and affinity for the ligand.
The expression and secretion of NPY and the expression of NPY-R isoforms are controlled by a very wide range of agents, acting in an endocrine and/or paracrine fashion. NPY and NPY-Rs appear to be strongly involved in the control of eating behavior; their expression is modulated by changes of food intake and energy balance and is disrupted in several animal models of obesity and diabetes. Moreover, the hypothalamic NPY system appears to integrate signals of energy balance in the modulation of the reproductive axis. Agents that stimulate their expression include activators of intracellular signalling pathways (protein kinase A and C), classical neurotransmitters, steroid and peptide hormones and growth factors, while other agents (leptin, insulin and retinoic acid) have been shown to be inhibitory. Interestingly, some agents, like retinoic acid, have been shown to modulate the expression of both NPY and NPY-Rs in the same direction, thus providing a fine mechanism for the tuning of the system.
The regulation of NPY/NPY-R
expression and function appears to be part of a complex system controlling
multiple physiological functions, and its disruption might be relevant in the
pathophysiology of disease states such as obesity.
Back to top]
Structural/Functional Aspects of ES-62 – A Secreted
Immunomodulatory Phosphorylcholine-Containing Filarial Nematode Glycoprotein
W. Harnett, M. M.
Harnett and O. Byron
ES-62 is a major secreted
glycoprotein of the rodent filarial nematode Acanthocheilonema viteae and
homologue of molecules found in filarial nematodes which parasitise humans. The
molecule consists of a tetramer of apparently identical monomers of ~62 kDa
which we have shown by sedimentation equilibrium analytical ultracentrifugation
to strongly associate. ES-62 is one of several filarial nematode proteins to
contain the unusual posttranslational modification of phosphorylcholine (PC)
addition. Specifically, we have found that PC is attached to one of three
distinct N-type glycans we have characterised on the molecule. The amino acid
sequence of ES-62 shows 37–39% identity with a family of 6 other proteins, some
of which have been predicted to be amino- or carboxy-peptidases. We have also
found that ES-62 is able to interact with a number of cells of the immune
system, specifically B- and Tlymphocytes, macrophages and dendritic cells.
Lymphocytes exposed to ES-62 in vitro or in vivo are less able to proliferate
in response to ligation via the antigen receptor. Peritoneal macrophages
pre-exposed to the molecule are less able to produce the cytokines IL-12, IL-6
and TNF-a following subsequent
incubation with the classical stimulators IFNg
and LPS. Dendritic cells allowed to mature in the presence of ES-62 acquire a
phenotype, which allows them to induce anti-inflammatory “TH2-type” responses.
With respect to immunomodulation, the PC moiety of the parasite molecule
appears to be predominantly responsible for the effects on lymphocyte
proliferation at least and we have also found that its removal converts the
murine IgG antibody response to ES-62 from solely IgG1 to mixed IgG1/IgG2a.
ES-62 appears to interact with cells of the immune system in a PC-dependent
manner and, at least in part, via a molecule of ~82 kDa. Studies of the
interaction in lymphocytes show that it is associated with activation of
certain signal transduction molecules including a number of protein tyrosine
kinases and mitogen activated protein kinases (MAPkinases). Although such
activation is insufficient to induce proliferation, it serves to almost
completely desensitise the cells to antigenreceptor ligation-induced activation
of the phosphoinositide 3-kinase (PI-3-kinase) and Ras/MAPkinase pathways, events
critical for lymphocyte proliferation. Such desensitisation reflects
ES-62-primed recruitment of a number of negative regulators of these pathways,
such as the phosphatases SHP-1 and Pac-1.
Back to top] Rapid Translation System
(RTS): A Promising Alternative for Recombinant Protein Production
J.-M. Betton
Rapid Translation System (RTS) is
a cell-free protein production system employing an enhanced Escherichia coli
lysate to perform coupled in vitro transcription-translation reactions. A
continuous supply of energy substrates, nucleotides and amino acids combined
with the removal of by-products guarantees a high yield of protein production.
The gene to express is either cloned into a plasmid vector or introduced as a
PCR product amenable to automation. The main property of this alternative
system to cellular expression systems is its open design allowing direct
manipulation of the reaction conditions and applications that are impossible or
difficult in cell-based systems. RTS offers new promising possibilities in the
postgenomic era.