trans

94 ANTIGENIC VARIATION
glycans are located toward the carboxy terminus,
they are inaccessible to lectins added to
suspensions of intact trypanosomes. In some
cases, living trypanosomes covered with a
specific VSG are resistant to trypsinization,
whereas the solubilized VSG is susceptible. This
suggests that even a small protein like trypsin
has problems penetrating into the deeper
regions of the coat. Thirdly, there have been
several studies in which panels of monoclonal
antibodies were generated against purified
VSGs. Only a small proportion of these antibodies
react with the surface of living trypanosomes.
This is consistent with the dense VSG packing,
which results in the display of only a small
area of the molecule on the trypanosome surface,
which is also consistent with the crystal
structure determined for the amino-terminal
domains of two VSGs (see below).
The VSG has no known enzymatic or receptor
function. The prevailing view is that its role
is simply to form a physical barrier that can be
exchanged, in a small fraction of the population,
as the immune response develops to
each VSG in turn. This barrier may have two
specific roles. It may prevent antibodies to
invariant or less variant membrane proteins
from reaching their targets on living cells.
Secondly, antibodies against VSG will not cause
complement-mediated lysis because complement
deposition cannot progress to membrane
insertion of the C9 complex. Whether
the VSG coat has a role in sequestering parasites
at different sites within the infected host
has been debated but little studied. Because of
the extent of variation, there could be fortuitous
or intentional sequestering of some trypanosomes
in specific organs. Trypanosomes
do not only circulate in the main vasculature,
so specific retention in the tissues, or the
invasion of specific VSG subtypes into the
brain, are issues that could be relevant to
pathogenesis.
VSG structure
The amino acid sequences of many VSGs have
been deduced from the sequences of the
corresponding genes. One VSG was sequenced
at both the protein and DNA level, which
incidentally proved that trypanosomes use
the ‘universal’ genetic code. Partial protein
sequencing of several VSGs identified key features
of the amino- and carboxy-terminal signal
sequences that are cleaved immediately
post-translationally, the carboxy-terminal signal
being replaced by the GPI anchor. Most
VSGs exist as non-covalently-linked homodimers,
but there are known exceptions, and
maybe more that are undiscovered, that are
either disulfide-linked or capable of forming
higher oligomers in solution. The VSGs of
T. brucei consist of an amino-terminal domain
linked to one or two carboxy-terminal subdomains
via a ‘hinge’ region that is exquisitely
sensitive to proteolytic cleavage and whose
presumed flexibility prevented crystallization
of intact VSG. Consequently, the crystal structures
of two amino-terminal domains were
determined some time ago (Figure 5.2), but
the structure of the carboxy-terminal domain
is unknown.
Although only a small area of the VSG is
accessible in the surface coat, amino acid
sequence variation occurs throughout the
molecule. If the extraordinarily conserved
carboxy-terminal GPI anchor signal sequence
is disregarded, the range of sequence variation
among VSGs is such that some sequences
would be difficult to recognize as members of
the family. However, known T. brucei VSGs can
be grouped into several classes, based primarily
on their GPI signal sequences and spacing
of conserved cysteine residues (Figure 5.3).
Despite the wide range of primary sequence
variation, VSG amino-terminal domains are
predicted to fold into similar three-dimensional
MOLECULAR BIOLOGY