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 In: Intrinsically Disordered Proteins

MYELIN BASIC PROTEIN

No part of this digital document may be reproduced, stored in a retrieval system or transmitted in any form or
by any means. The publisher has taken reasonable care in the preparation of this digital document, but makes no
expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No
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rendering legal, medical or any other professional services.
 INTRINSICALLY DISORDERED PROTEINS
Series Editors: Vladimir N. N. Uversky and A. K. Dunker

Measles Virus Nucleoprotein

Sonia Longhi
2008. ISBN-13: 978-1-60021-629-9

Myelin Basic Protein

Joan M. Boggs
2008. ISBN 978-1-60456-699-4
 In: Intrinsically Disordered Proteins

MYELIN BASIC PROTEIN

JOAN M. BOGGS
EDITOR

Nova Science Publishers, Inc.

New York
Copyright © 2008 by Nova Science Publishers, Inc.

All rights reserved. No part of this book may be reproduced, stored in a retrieval system or
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LIBRARY OF CONGRESS CATALOGING-IN-PUBLICATION DATA

Myelin basic protein / Joan M. Boggs (editor).
p. ; cm.
Includes bibliographical references and index.
ISBN 978-1-60876-247-7 (E-Book)
1. Myelin basic protein. 2. Myelin sheath. I. Boggs, Joan M.
[DNLM: 1. Myelin Basic Proteins--physiology. 2. Multiple Sclerosis--physiopathology. QU 55.7
M996 2008]
QP552.M88.M94 2008
572'.633--dc22 2008017062

Published by Nova Science Publishers, Inc. New York

 CONTENTS

Preface vii
Chapter I The Properties and Functions of the Golli Myelin Basic Proteins 1
Anthony T. Campagnoni and Celia W. Campagnoni
Chapter II Posttranslational Modifications of Myelin Basic Proteins 19
Robert Zand
Chapter III Deimination of Myelin Basic Protein by PAD Enzymes, and Their
Role in Multiple Sclerosis 31
Fabrizio G. Mastronardi and Mario A. Moscarello
Chapter IV Myelin Basic Protein-Mediated Immunopathogenesis in Multiple
Sclerosis and EAE 51
Qingyong Ji and Joan Goverman
Chapter V A Structural Perspective of Peptides from Myelin Basic Protein 87
Maria Katsara, Paul A. Ramsland, Theodore Tselios,
John Matsoukas and Vasso Apostolopoulos
Chapter VI Interactions of the 18.5 kDa Myelin Basic Protein with Lipid
Bilayers: Studies by Electron Paramagnetic Resonance
Spectroscopy and Implications for Generation of Autoimmunity in
Multiple Sclerosis 105
Joan M. Boggs, Ian R. Bates, Abdiwahab A. Musse
and George Harauz
Chapter VII Insights into the Interaction of Myelin Basic Protein with
Microtubules 127
Mauricio R. Galiano, Cecilia Lopez Sambrooks and
Marta E. Hallak
Chapter VIII Myelin Basic Protein Interactions with Actin and Tubulin In Vitro:
Binding, Assembly, and Regulation 149
Joan M. Boggs
vi Contents

Chapter IX Molecular Modelling of the Interaction of Myelin Basic Protein

Peptides with Signalling Proteins and Effects of Post-Translational
Modifications 169
Eugenia Polverini
Chapter X Structure and Dynamics of the Myelin Basic Protein Family by
Solution and Solid-State NMR 197
George Harauz and Vladimir Ladizhansky
Index 233
 PREFACE

The compact myelin sheath formed around nerve axons speeds up nerve conduction and
also nurtures the axon. Destruction of this sheath in demyelinating diseases such as multiple
sclerosis (MS) results in nerve conduction failure and neurodegeneration. Myelin basic
protein (MBP) is the second most abundant protein of central nervous system (CNS) myelin
(after the proteolipid protein), representing about 30% of the total myelin protein and about
10% of myelin by weight. It is also present in peripheral nervous system (PNS) myelin but as
a lower percentage of the total protein. In the CNS, myelin is formed by oligodendrocytes
which extend membrane processes that wrap around the axon (Figure).

Figure. Diagram of the myelin sheath formed by an oligodendrocyte process around a nerve axon. The
extracellular surfaces are apposed at the intraperiod line (thin black line) in compact myelin. MBP
mediates adhesion at the cytosolic surfaces forming the major dense line (heavy red line). The
paranodal loops, outer loop and inner loop contain cytosol, including cytoskeletal proteins. The radial
component consists of a series of tight junctions and may also contain actin, and tubulin (see Chapter
VIII). The nerve axon is bare at the node of Ranvier. Adapted from Boggs et al. (2008) and republished
with permission from Elsevier. I thank George Harauz for providing an earlier version of this figure.
viii Joan M. Boggs

MBP binds to negatively-charged lipids on the cytosolic surfaces of the processes and is
responsible for adhesion of these surfaces of myelin in the CNS, thus forming the major dense
line observed by electron microscopy. It is thus a structural protein that has been shown to be
essential for formation of compact CNS myelin; a naturally occurring shiverer mutant mouse,
which has a deletion of the major part of the gene encoding MBP, produces only small
amounts of uncompacted myelin. However, MBP is not essential for formation of PNS
myelin, due to the presence of other proteins specific to PNS myelin, that may compensate for
its absence.
MBP was first discovered in the 1960s by Dr. Marian Kies as a result of efforts to
determine the factor from brain which induced experimental allergic encephalomyelitis
(EAE) (Kies et al., 1961; Laatsch et al., 1962). The most abundant 18.5 kDa isoforms from
bovine and human brains were sequenced in 1971 by Eylar et al. (1971) and Carnegie (1971),
respectively. MBP is now known to be the product of a gene which has three different
transcription start sites and yields two major families of proteins, the “classic” MBP family,
which is expressed only in myelin and myelin-producing cells, and the golli proteins, which
are also expressed in other cells, including immune tissue. The MBP gene and the functions
of the golli proteins are described in Chapter I by Campagnoni and Campagnoni. Classic
MBP also exists as a number of size isoforms due to differential splicing, and can be post-
translationally modified in a number of ways, resulting in a diverse family of proteins (see
Chapter II by Zand, and Chapter III by Mastronardi and Moscarello).
Although it is now known that other proteins from myelin can also induce EAE, and that
determinant spreading occurs during autoimmune responses, the encephalitogenic properties
of MBP have attracted wide interest in attempts to understand its role in the demyelinating
disease multiple sclerosis (MS). Both T cell and B cell-mediated immune responses to MBP
occur in MS, and its antigenic epitopes have been characterized (see Chapter IV by Ji and
Goverman). The structures of immunogenic MBP peptides complexed with class II MHC
proteins have been determined in order to aid in the design of synthetic peptides which will be
useful for suppression of this immune response (see Chapter V by Katsara, Ramsland,
Tselios, Matsoukas, and Apostolopoulos). In addition to inducing autoimmune response, a
particular variant of MBP, with significantly reduced net positive charge, may be further
involved in demyelination in MS. This charge isomer, in which a number of arginines are
deiminated by the enzyme peptidyl arginine deiminase (PAD) to give uncharged citrulline
residues, occurs normally in higher amounts in children than in adults. However, both it and
the PAD enzyme are found in increased amounts in brains of adults with MS (see chapter III).
This less-charged MBP variant has decreased ability to cause adhesion of negatively-charged
lipid membranes, which may destabilize myelin. It may also elicit an increased immune
response, as discussed below.
Despite the early amino acid sequencing of MBP and the characterization of its gene
structure, attempts to determine its tertiary structure by crystallography, solution NMR
spectroscopy, and other methods, have been unsuccessful. The reason is the large number of
charged residues throughout the protein’s sequence, and its low overall hydrophobicity,
which maximize intramolecular electrostatic repulsion, resulting in an extended, natively-
unfolded structure (Harauz et al., 2004). Such proteins have sufficient flexibility to bind to
various charged surfaces and ligands, and to acquire whatever local conformation is necessary
to optimize specific binding to several different targets (Dyson and Wright, 2002). Site-
directed spin labeling and EPR spectroscopy are now being applied to determine the structure
 Preface ix

of MBP, when bound to lipids and other proteins, from the environment of its spin-labeled
residues (see Chapter VI by Boggs, Bates, Musse, and Harauz). These studies have revealed
that when MBP is bound to a lipid surface, an immunodominant epitope of MBP forms an
amphipathic alpha-helix with its hydrophobic surface embedded in the bilayer, and its
hydrophilic surface exposed. They further showed that deimination results in dissociation of
much of the C-terminal half of MBP from the membrane surface, shortens the length of the
amphipathic alpha-helix formed by the immunodominant epitope, and increases its
susceptibility to proteolytic digestion. Greater exposure to proteolytic enzymes can cause
release of this immunodominant epitope of MBP, which may initiate or sustain immune
response to this epitope, as discussed in Chapter VI.
Studies of other intrinsically disordered proteins have shown that they are often
multifunctional regulatory proteins (Tompa et al., 2005) involved in multiple interactions,
which may integrate hubs of various biological activities (Uversky et al., 2005; Patil and
Nakamura, 2006). In this regard, MBP also interacts with several other proteins in addition to
binding to negatively-charged lipids, and has also been suggested to be a multifunctional
protein (Boggs, 2006). It binds to actin, tubulin, tropomyosin, calmodulin, and clathrin and
assembles microfilaments, microtubules, and clathrin baskets in vitro. MBP has been shown
to be one of only two proteins isolated from the brain with the ability to stabilize
microtubules from depolymerizing (STOP activity) in the cold, and also has this activity in
oligodendrocytes (see Chapter VII by Galiano, Sambrooks, and Hallak). It is also able to
bind bundles of actin filaments to lipid surfaces and may be able to serve as a membrane
actin-binding protein (see Chapter VIII by Boggs). MBP’s binding to actin and tubulin can
be regulated by Ca2+-calmodulin, and is influenced by its various post-translational
modifications. The MBP-mediated binding of actin filaments to the membrane can also be
regulated by changes in membrane surface potential, which could be achieved through local
changes in lipid composition during signal transduction. Chapters VII and VIII describe
studies suggesting that MBP is involved in transmitting extracellular signals received on the
oligodendrocyte surface to the cytoskeleton.
In addition to interactions with actin and tubulin, which may be primarily electrostatic,
MBP has a domain that has been predicted to be a PXXP SH3-target consensus sequence
(Moscarello, 1997). This domain has recently been shown to form a poly-proline helix, and
to bind SH3 domains of several proteins (see Chapter IX by Polverini). The interactions of
MBP with both Ca2+-calmodulin and the SH3 domain of Fyn tyrosine kinase, and the effects
of post-translational modifications to MBP on these interactions, have been modeled in silico
and are described in Chapter IX. Since the binding of MBP to different ligands can induce or
stabilize a particular conformation, its structure when bound to lipids, Ca2+-calmodulin, and
other proteins is now being studied by solution and solid-state NMR spectroscopy (see
Chapter X by Harauz and Ladizhansky). Further study of the structure of members of the
MBP family when bound to different ligands and in different environments will help to
understand its role in signaling in oligodendrocytes and myelin, and its involvement in the
pathogenesis of MS.
I wish to take this opportunity to thank all of the authors for their chapters in this book
and to Dr. George Harauz in particular, for helpful advice during its preparation. I also
x Joan M. Boggs

apologize to those whose work could not be included or was improperly cited because of
space and time limitations. I hope that this book will be a valuable contribution to the study
of myelin basic protein and myelin.

Joan M. Boggs, Editor

REFERENCES
Boggs, J.M. (2006). Myelin basic protein: a multifunctional protein. Cell. Mol. Life Sci, 63,
1945-1961.
Boggs, J.M., Gao, W., and Hirahara, Y. (2008). Myelin glycosphingolipids,
galactosylceramide and sulfatide, participate in carbohydrate-carbohydrate interactions
between apposed membranes and may form glycosynapses between oligodendrocyte or
myelin membranes. Biochim. Biophys. Acta, 1780, 445-455.
Carnegie, P.R. (1971). Amino acid sequence of the encephalitogenic basic protein from
human myelin. Biochem. J, 123, 57-67.
Dyson, H.J., and Wright, P.E. (2002). Coupling of folding and binding for unstructured
proteins. Curr. Opin. Struct. Biol, 12, 54-60.
Eylar, E.H., Brostoff, S., Hashim, G., Caccam, J., and Burnett, P. (1971). Basic A1 protein of
the myelin membrane: The complete amino acid sequence. J. Biol. Chem, 246, 5770-
5784.
Harauz, G., Ishiyama, N., Hill, C.M.D., Bates, I.R., Libich, D.S., and Farès, C. (2004).
Myelin basic protein - diverse conformational states of an intrinsically unstructured
protein and its roles in myelin assembly and multiple sclerosis. Micron, 35, 503-542.
Kies, M.W., Murphy, J.B., and Alvord, E.C., Jr. (1961). Studies of the encephalitogenic
factor in guinea pig central nervous sytems. In J. Folch-Pi (Ed.), Chemical Pathology of
the Nervous System (pp. 197). Elmsford, NY, USA: Pergamon Press.
Laatsch, R.H., Kies, M.W., Gordon, S., and Alvord, E.C., Jr. (1962). The encephalitogenic
activity of myelin isolated by ultracentrifugation. J. Exp. Med, 115, 77-88.
Moscarello, M.A. (1997). Myelin basic protein, the 'executive' molecule of the myelin
membrane. In B.H.J. Juurlink, R.M. Devon, J.R. Doucette, A. J. Nazarali, D.J. Schreyer,
V.M.K. Verge (Eds.), Cell Biology and Pathology of Myelin: Evolving Biological
Concepts and Therapeutic Approaches (pp. 13-25). New York, NY, USA: Plenum Press.
Patil, A., and Nakamura, H. (2006). Disordered domains and high surface charge confer hubs
with the ability to interact with multiple proteins in interactions networks. FEBS Lett,
580, 2041-2045.
Tompa, P., Szasz, C., and Buday, L. (2005). Structural disorder throws new light on
moonlighting. Trends Biochem. Sci, 30, 484-489.
Uversky, V.N., Oldfield, C.J., and Dunker, A.K. (2005). Showing your ID: intrinsic disorder
as an ID for recognition, regulation and cell signaling. J. Mol. Recog, 18, 343-384.
In: Myelin Basic Protein ISBN: 978-1-60456-699-4
Editor: Joan M. Boggs © 2008 Nova Science Publishers, Inc.

Chapter I

THE PROPERTIES AND FUNCTIONS OF THE GOLLI

MYELIN BASIC PROTEINS

Anthony T. Campagnoni* and Celia W. Campagnoni*

ABSTRACT
Like the classic myelin basic proteins, the golli-MBPs are intrinsically unstructured
protein products of the MBP gene. They are expressed in numerous cell types throughout
the immune and nervous systems, and their function(s) are beginning to be understood.
Since their discovery over a decade ago a number of approaches have been taken to
elucidate their structure, including searches for binding partners, selective ablation of
their expression in knock-out mice, and their overexpression in transgenic animals and in
cell models. They appear to have an, as yet, undetermined role in the nucleus, but they
have now been clearly shown to regulate Ca++ homeostasis in T-cells through modulation
of CRAC channels, and in oligodendrocytes through voltage-gated Ca++ channels as well
as through store-operated and ligand-gated Ca++ channels. Thus, they appear to play a
significant role in oligodendrocyte development and in Ca++-dependent processes, such as
process extension/retraction and migration, as well as T-cell activation. Several studies
have also shown that expression of the golli-MBPs is altered in a number of human and
animal neuropathological conditions, providing further evidence for their importance in
cellular function.

*
Semel Institute for Neuroscience and Human Behavior, Geffen School of Medicine, University of California at
Los Angeles, Neuroscience Research Building, 635 Charles Young Drive, Los Angeles, CA 90095-7332.
Telephone: 1-310-825-5006; Fax: 1-310-206-5050; E-mail: [email protected];
[email protected]
2 Anthony T. Campagnoni and Celia W. Campagnoni

INTRODUCTION
About twenty years ago reports of the isolation of unexpected alternatively spliced
products of the MBP gene began to appear in the literature (Newman et al., 1987;
Campagnoni et al., 1993; Zelenika et al., 1993), but a clear relationship of these products to
the MBP gene was not firmly established until the entire gene structure was elucidated
(Campagnoni et al., 1993). The publication of the revised MBP gene structure and the golli
products of the gene was met initially with some confusion and also some skepticism.
Relative to the classic MBPs, among the most abundant proteins in the nervous system, the
levels of the golli proteins seemed too low to be of real significance. Furthermore, the
introduction of a second family of proteins that was immunologically similar to the known
MBPs and expressed in both the immune and nervous systems, raised questions about prior
concepts of “tolerance” in EAE, which had been studied for decades as a model for the
inflammatory component of MS (Huseby and Goverman, 2000; Maverakis et al., 2000;
2003). Furthermore, the exact relationship between the classic MBPs, the golli-MBPs and
their relationship to the structure of the MBP gene was confusing to some because the golli
transcription unit was initially referred to as “overlapping” the classic MBP gene, when, in
fact, the entire complex should be viewed as a single gene (see section on gene structure).
In the years since these proteins were identified, the importance of the golli proteins to
the biology of T-cells and oligodendrocytes has become more clearly established. We are
now beginning to understand the function of the golli proteins in Ca++ homeostasis in these
cells and we have identified other potential roles for these proteins in the cell deserving of
further investigation. In this review we summarize findings about the structural, molecular
and cell biological properties of the golli proteins and our present understanding of the
function of these proteins in T-cells and in oligodendrocytes.

The MBP gene encodes the “classic” and golli family of proteins

The structure of the MBP gene in mouse and human and their major golli splice products
are diagrammed in Figure 1. The mouse MBP gene is ~ 105Kb in length (Campagnoni et al.,
1993) and the human gene is ~ 180 Kb (Pribyl et al., 1993). The MBP gene contains three
independent promoters, and mRNA products from all three transcription initiation sites have
been identified in the mouse (Campagnoni et al., 1993; Fritz and Kalvakolanu, 1995; Zelenika
et al., 1993; Kitamura et al., 1990) and from the first and third sites in the human (Roth et al.,
1987; Pribyl et al, 1993; Grima et al., 1994; Tosic et al., 2002). The presence of exon 4 and
the second transcription initiation site in the human gene is inferred.
The classic MBP mRNAs are derived from transcription start sites 2 and 3, and constitute
major protein constituents of the myelin membrane. The most downstream promoter,
governing tss3, is the strongest of the three promoters and is very active in oligodendrocytes.
As such they encode some of the most abundant proteins in the brain and they are expressed
almost exclusively in myelin-forming cells, although expression of low levels of classic
MBPs has been reported in the immune system (Liu et al., 2001).
 The Properties and Functions of the Golli Myelin Basic Proteins 3

Golli-MBP transcription start site classic MBP transcription start sites

tss1 tss2 tss3
C

ABC AB
Exons 1 2 3 4 5 6 7 8 9 10 11

mouse golli splicing (tss1) human golli splicing (tss1) classic MBP splicing
(tss2 & tss3)

1 2 3 5 1 2 3 5 classic
BG21 mRNA A B C HOG5 mRNA A B C MBP mRNA family
1 2 3 5 7 8 11 1 2 3 5 7 8 9 1011
J37 mRNA A B HOG7 mRNA A B B

1 2 3 7 8 11 1 2 3 5 7 8 10 11
TP8 mRNA NHOG1 mRNA A B

(133 aa) (57 aa) (133 aa) (59 aa)

MBP MBP
BG21 protein golli domain HOG5 protein golli domain
domain domain

(133 aa) (117 aa) (133 aa) (171 aa)

J37 protein golli domain MBP domain HOG7 protein golli domain MBP domain

(47aa) (22 aa) (133 aa) (161 aa)

TP8 protein golli domain NHOG1 protein golli domain MBP domain

Figure 1. Diagram of the myelin basic protein gene showing the generation of the major golli-MBP
products in human and mouse. The fourth exon has not been demonstrated in the human, but is inferred
by analogy with the mouse. (See Fig. 1 in Chapter X for correspondence of former exon numbering of
classic MBP with new numbering of all exons in total MBP gene).

The second family of proteins encoded by the gene is the golli proteins. The golli
mRNAs are generated from the first transcription start site and they are expressed more
ubiquitously than the classic MBPs. In contrast to the classic MBPs, they are expressed at
similar levels in the thymus, spleen and brain (Feng et al., 2004). Many cell types, e.g.,
thymocytes, T-cells, B-cells and macrophages, as well as neurons and oligodendrocytes in the
nervous system express the golli products.
The golli mRNAs from mouse and human all contain exons 1-3, and these encode a 133
aa golli domain with related primary sequences. Figure 1 shows the major golli cDNA
products that have been cloned from mouse brain and human brain. Mouse golli TP8 and its
unpublished human homologue are minor products. While they encode the golli peptide, they
contain no classic MBP sequences, since the classic MBP exons are read out of frame. The
major products of the golli gene also express MBP epitopes. Thus BG21 and its human
ortholog, HOG5, contain the golli domain immediately upstream (i.e. on the N-terminal side)
of the first 57 (mouse) or 59 (human) amino acids of classic MBP (Figure 2A). The mouse
J37 consists of the 133 aa golli domain fused to a small MBP that does not correspond to one
of the classic MBPs, however the human HOG 7 consists of the golli domain fused in frame
to the human 18.5 kDa MBP. In mouse, BG21 and J37 are the major isoforms expressed
throughout development; but in human brain the major isoforms change during development.
For example, in fetal human brain the principal golli mRNAs appear to be HOG 5 and
NHOG1, a golli isoform consisting of the golli domain fused to a 17 kDa classic MBP
sequence (see Figure 2B).
4 Anthony T. Campagnoni and Celia W. Campagnoni

Figure 2. (A) Sequence comparison of the BG21 (mouse) and HOG5 (human) golli orthologs. (B)
Sequence comparison of the mouse J37 and human HOG7 & NHOG1 golli isoforms, which contain
longer classic MBP sequences than either BG21 or HOG5. (C) Illustration of identified and potentially
significant residues and domains in the BG21 molecule. Potential phosphorylation sites are noted by
speckled circles. Other sites and domains are shown.

In the more mature human brain the principal isoforms are HOG5 and HOG7. This
developmental shift from NHOG to HOG 7 reflects the exon splicing pattern shift that
normally occurs within the human classic MBPs with development (Roth et al 1987; Pribyl et
al., 1996a). HOG 5/HOG 7 and HOG 5/NHOG1 constitute about 80% of the transcripts
found in human tissue, although numerous other splice variants also have been identified
(Pribyl et al., 1996a).
 The Properties and Functions of the Golli Myelin Basic Proteins 5

Features of the primary and higher ordered structure of the golli-MBPs

Unlike the classic MBPs, the golli proteins do not appear to be normal components of the
myelin sheath, but are localized within the nuclei, cell bodies and primary processes of
oligodendrocytes and neurons (Landry et al., 1996; Paez et al. 2007). As indicated, they are
more ubiquitously expressed throughout the nervous system and the immune system than are
the classic MBPs, which are primarily products of myelin forming OLs, and they have
different developmental patterns of expression (Campagnoni et al., 1993; Pribyl et al., 1993;
Landry et. al., 1996;1997). This, and their non-inclusion in the myelin sheath, suggested that
they had some other biological function than the classic MBPs (Campagnoni & Skoff, 2001).
A clue to the function of proteins often comes from the physical properties of, and the
presence of consensus sequences and domains in, the molecule. The J37 and HOG 7
isoforms contain significantly longer classic MBP sequences than BG21 or HOG 5; and, as
might be expected, the pIs of the golli isoforms mirror the amount of (highly basic) classic
MBP sequence found within the molecule. For example, BG21 and HOG 5 have pIs of ~6
and J37 and HOG7 have pIs from 9.6-9.8. All these golli isoforms have mean net charges
and mean net hydrophobicity values that would categorize them as potential intrinsically
unstructured proteins (Ahmed et al., 2007).

Primary sequence
A survey of the primary sequences of the mouse golli proteins predicts calmodulin-
binding three calmodulin-binding motifs in J37, two in BG21, and four in HOG7 (Polverini et
al., 2004) (see Chapter IX and see Figure 2C for BG21). Experimentally, both recombinant
J37 and BG21 bind calmodulin at a 1:1 ratio in the presence of calcium but the association
appears to be weak, since even at high ratios of calmodulin: BG21, a significant amount of
unbound BG21 remained. Titration curves of BG21 with calmodulin monitored either by
fluorescence intensity of the single tryptophan at 346 or by fluorescence anisotrophy did not
plateau so it was impossible to calculate a dissociation constant (Kaur et al., 2003; Bamm et
al., 2007).
There is an essential myristoylation motif at the amino terminus of the golli proteins.
Elimination of this site by site-directed mutation of G2 to A essentially eliminates golli
function as a Ca++ regulator in both T cells and oligodendrocyte cell lines (Feng et al., 2006;
Paez et al., 2007). Chemical confirmation of the presence of the myristoylated glycine
residue has been obtained from LC-MS analysis (C. Campagnoni and K. Faull, unpublished)
Golli proteins are phosphorylated in vivo (Feng et al. 2004). BG21 transfected into
Jurkat T cells grown in the presence of 32P orthophosphate was immunoprecipitated as a
phosphoprotein and the amount of phosphorylation increased when the cells were activated
with the PKC stimulator, PMA. However, it is not yet clear what kinases or signaling
pathways might be involved. Feng et al (2004) investigated two kinases and found that while
both BG21 and J37 could be phosphorylated in vitro by PKC, only J37 could be
phosphorylated by Erk 2 (MAPK). The 133 aa golli-specific peptide could be phosphorylated
by neither kinase. As shown in Figure 2C, there are at least 5 potential PKC sites on the
molecule, i.e. S 5 and T106 in the golli domain and S136, 141, 188 in the MBP region common to
both BG21 and J37.
6 Anthony T. Campagnoni and Celia W. Campagnoni

Higher ordered structure of golli proteins

Harauz and his colleagues have extensively characterized the mouse recombinant golli
proteins. While classic MBPs aggregate phospholipid vesicles, J37 (a golli isoform with
significant classic MBP sequence) had no effect on a myelin-like preparation. Circular
dichroism studies, however, showed that BG21 and J37, which are largely random coils in
aqueous solution, each acquired more organized secondary structures in the presence of
ganglioside GM1 or phospholipids, such as PI(4)P (Kaur et al., 2003; Bamm et al., 2007).
Heteronuclear NMR measurements in 0.1M KCl confirmed that BG21 has little ordered
secondary structure in solution and revealed that residues S5-T69 were unusually flexible even
for IUPs (see Chapter X). This region was postulated to be a candidate for protein-protein
interactions, and another smaller mobile segment, A126 to G129 was postulated to be a hinge
(Ahmed et al., 2007) (see Figure 2C).
The analyses of the primary and higher ordered structures of the protein provided
evidence for a critical myristoylation site, suggesting a need for association of golli with a
membrane in order for it to be functional. They also suggested a potential ability of golli to
perform its functions through interactions with other proteins, a characteristic common to
IUPs. From what is now known about the cell biology of golli proteins, these interactions
could occur in complexes at the plasma membrane or in the nucleus (see below).

Nuclear localization sequence in golli proteins

There is evidence that sequences found within exon 6 of the (golli) MBP gene (please
refer to Figure 1 for exon numbering) are responsible for targeting certain classic MBP
isoforms, such as the 17kDa and 21.5kDa MBPs, to oligodendrocyte nuclei early in postnatal
brain (Pedraza et al., 1997). In most cells, golli immunocytochemical analysis suggests that
golli proteins are localized in both the nucleus and cytoplasm/processes. Interestingly, in
certain cell types the golli MBPs appear to undergo rather dramatic subcellular localization
shifts during development (Landry et al., 1996). This is particularly evident in cerebellar
granular cells. During development, immature granule cells migrate from the outer layers of
the cerebellum to a deeper layer where they form the internal granule cell layer in the mature
cerebellum. Accompanying this transition is a significant shift of the localization of golli
from the cell body and processes of these cells to the nucleus. Transfection studies with
mutated golli cDNAs have identified a nuclear targeting element within a 36 amino acid
region of the golli proteins, M134-I169, located in the MBP domain (Reyes and Campagnoni,
2002). The BG21 isoform of golli does not contain sequences derived from exon 6 (see
Figure 1) found in the 17kDa and 21.5kDa classic MBPs, so its translocation to the nucleus
cannot be attributed to the same NLS as that of the classic MBPs. Neither the regulation of
nuclear targeting of golli proteins nor the function(s) of golli proteins in the nucleus have
been resolved.

Approaches to defining the biological roles of golli proteins in cells

Several approaches have been used to identify the cellular function(s) of the golli proteins
in the immune and nervous systems. These have included a search for binding partners using
the yeast two-hybrid system with the golli domain as a “bait” (Fernandes et al., 2004), loss-
 The Properties and Functions of the Golli Myelin Basic Proteins 7

of-function analyses through ablation of golli expression in knock-out (KO) mice (Jacobs et
al., 2005; Feng et al., 2006) and gain-of-function analyses (a) through transfection of golli
isoforms into cells in vitro (Reyes and Campagnoni, 2002; Paez et al., 2007) and (b) through
cell-specific targeting of golli into oligodendrocytes in transgenic mice (Reyes et al., 2003;
Martin et al., 2007).

Golli proteins bind to nuclear proteins involved in gene transcription

Fernandes et al. (2004) conducted a yeast two-hybrid screen of a rat PC12 library using
the 133 aa golli domain as “bait”. From this library a clone was isolated that encoded a golli-
interacting protein (GIP) with a predicted molecular weight of 25kDa; and the rat clone was
used to isolate the mouse homolog from a mouse oligodendrocyte library.
Immunocytochemical analysis indicated that GIP was co-expressed with golli proteins in a
wide variety of cells and that it was localized predominantly in the nuclei of these cells.
Immunoprecipitation studies showed that GIP interacted with nuclear LIM interactor (NLI), a
nuclear protein known to associate with LIM transcription factors, as well as the golli
proteins; and that, in fact, all three could form a trimolecular complex with GIP serving as the
intermediary.
GIP is identical to SCP-1 of a series of small carboxyl-terminal domain (CTD)
phosphatases described by Gill and coworkers (Yeo et al., 2003; 2005). In eukaryotic cells,
the transcriptional activity of RNA polymerase II is modulated in part by the phosphorylation
status of S2 and S5 in a 26-52 tandem heptapeptide repeat at its carboxyl terminus. SCP-1
preferentially dephosphorylates S5 in the consensus sequence Y1S2P3T4S5P6S7. It co-
immunoprecipitates with a number of transcription factors, among them the RE-1 silencing
transcription factor (REST/NRSF) which is believed to be responsible for silencing the
transcription of neural genes in non-neural cells (Schoenherr and Anderson, 1995; Yeo et al.,
2005). SCP-1 appears to reduce transcription of the genes to which it is bound, perhaps by
slowing down clearance of transcription factors from the promoter (Thompson et al., 2006).
The existing information clearly indicates that golli proteins can bind to known proteins
in transcriptional complexes, but the mechanisms underlying its transport to the nucleus and
the specific gene activities it might be involved in regulating still remain unknown.

Golli proteins DECREASE Ca++ entry into T-cells upon activation of the cells
The nuclear-cytoplasmic shift of golli observed in neural cells suggested that golli
proteins might be involved in some aspect of intracellular signaling, since many signaling
molecules have been shown to shuttle between the cytoplasm or plasma membrane and the
nucleus. However, definite proof of a signaling role for golli-MBPs came from an
examination of the activation of T cells. These cells have been well studied because they are
easily stimulated and a good deal is now known about downstream signaling events after
activation of the T-cell receptor. For example, synthesis of IL2 is a hallmark of T cell
activation and the pathway from T cell receptor (TCR) engagement to the activation of the
IL2 promoter via AP-1, NFЌB and NFAT has been elucidated. Furthermore, TCR activation
also initiates a cascade of tyrosine kinase events that recruit PKCθ to the TCR and activates
phospholipase Cγ. Downstream of these events, the release of IP3 from phosphatidyl inositol
by PLC causes a release of Ca++ from internal stores, which, in turn, triggers an influx of Ca++
through calcium-release activated channels (CRAC) in the plasma membrane. Both IL2 gene
8 Anthony T. Campagnoni and Celia W. Campagnoni

transcription and CRAC activation can be triggered by activation of PKC with the phorbol
ester, PMA.
Feng and coworkers examined the role of golli proteins in T-cells extensively (Feng et
al., 2004; 2006). They found that BG21 is the major golli isoform expressed in T cells and
that when Jurkat T cells were transfected with BG21-GFP the protein behaved like an
intracellular signaling molecule. For example, immunofluorescence studies showed that upon
activation of the cells with PMA, the BG21-GFP fusion protein translocated from the
cytoplasm to the plasma membrane in a fashion identical to PKCθ. This was confirmed by
Western blots of membrane fractions, which also showed movement of BG21 from cytosolic
fractions to lipid raft fractions in sucrose density gradients after activation of the cells.
In vitro transfection of golli into Jurkat T cells inhibited IL-2 reporter gene transcription
upon TCR engagement (Feng et al., 2004). Although golli proteins possess several PKC
phosphorylation sites, Ser 136, 141, and 188, within the MBP domain common to both BG21
and J37, the inhibitory function of golli was independent of its PKC phosphorylation (Feng et
al., 2004) and resided in the golli domain alone (133aa). Subsequent studies showed that golli
acted negatively on T-cell receptor signaling by inhibiting store-depletion-induced Ca++ entry
into the T-cell through CRAC channels (Feng et al., 2006). Although phosphorylation of
these sites does not appear to be necessary for golli to modulate T-cell activation, they may
play another role unrelated to calcium uptake in these cells, possibly in the nucleus.

Golli proteins INCREASE Ca++ entry into oligodendrocytes upon activation of the cells
Golli proteins also have been shown to modulate Ca++ entry into oligodendrocytes,
although in an opposite fashion (Jacobs et al, 2005; Paez et al., 2007). In T-cells golli inhibits
Ca++ uptake upon stimulation of the T-cell receptor, but in oligodendrocytes it enhances Ca++
entry via voltage-gated channels under depolarizing conditions. These in vitro data were
obtained from a comparison of Ca++ changes in primary cultures of normal OLs vs. OLs in
which the golli products of the gene were selectively ablated. In these studies, the absence of
the golli proteins decreased Ca++ uptake when the cells were exposed to agents known to
induce Ca++ influx in OLs, e.g. high K+, AMPA, PMA and caffeine (Jacobs et al, 2005).
These results indicated that under normal circumstances golli proteins enhance Ca++ uptake
into OLs.
Modulation of intracellular Ca++ levels is important in a number of OL activities, such as
cell-cell communication (Simpson et al 1997), process extension (Yoo et al, 1999), migration
(Simpson & Armstrong 1999) and oligodendrocyte differentiation and myelination (Soliven,
2001). It has also been proposed that Ca++ surges may be involved in the signal for
myelination in a remyelinating animal model (Mateo Paz Soldan et al., 2003). Direct effects
of golli proteins on Ca++ mediated process extension (Paez et al., 2007) and migration (P.
Paez and A. Campagnoni, unpublished results) have now been shown.

Unique phenotypes of the golli KO and golli overexpressing mice

Elucidation of the function(s) of the golli proteins has been aided substantially by the
generation of mice in which the golli proteins were selectively ablated in all cells and tissues
(i.e., golli KO mice; Jacobs et al., 2005); and mice in which overexpression of the golli J37
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