trans

96 ANTIGENIC VARIATION
structures. The same is probably true of the
smaller carboxy-terminal subdomains, which
tend to have more-conserved sequences and
appear to be very tightly folded. Interestingly,
the few sequenced VSGs of T. congolense and
T. vivax are quite distinct from those of T. brucei
(Figure 5.3). They are smaller and lack any clear
carboxy-terminal domain, which is probably
relevant to assessing evolutionary relationships
among the various African trypanosomes.
VSG synthesis, processing, secretion
and turnover
Trypanosomes contain components of the
conventional eukaryotic signal recognition
machinery, and VSG contains an apparently
conventional amino-terminal signal sequence,
although there is published evidence that signal
sequences are not so exchangeable as one
might imagine. VSG is presumably synthesized
on ribosomes that are bound to the endoplasmic
reticulum, and the nascent polypeptide is
translocated into the lumen of the endoplasmic
reticulum, with co-translational cleavage of the
amino-terminal signal sequence. Cleavage of
the carboxy-terminal GPI signal sequence and
covalent coupling of the pre-assembled core
GPI anchor also appears to be a coupled and
immediately post-translational event, concomitant
with or prior to N-glycosylation. It has been
proposed that the secondary glycosylation of
the VSG-linked GPI anchor and N-linked glycans
represents a ‘space-filling’ function, determined
by the accessibility of the primary glycans
to further glycosylation by Golgi-resident
glycosyl-transferases, as VSG dimers traverse
the secretory pathway. In trypanosomes engineered
to express two VSGs simultaneously,
the patterns of N-glycosylation and GPI glycosylation
are almost identical to those found
when each VSG is expressed individually in its
normal cell background, which confirms that
the overall structures of the VSG homodimers
determine the extent and nature of the secondary
glycan modifications, not the cell background
in which they are expressed.
VSG half-life is more than 30 h (about 5 cell
generations), yet VSG is continually and rapidly
recycled, in the absence or presence of
specific antibody. It is not known whether a
mechanism exists for more rapid removal of
the ‘old’ VSG, during a VSG switch. The only
recognized site of endo- and exocytosis in trypanosomes
is the flagellar pocket, an invagination
at the exit point of the flagellum, which
accounts for a small part of the total surface
membrane area but turns over at a high rate.
Certain ‘cell-surface’ proteins appear to be
specifically retained in the flagellar pocket by
a mechanism that may depend on a unique
glycosylation pattern recognized by hypothetical
trans-membrane receptor lectins in the
flagellar pocket. In the context of antigenic variation,
the flagellar-pocket retention of the heterodimeric
transferrin receptor is intriguing, as
it is also GPI anchored, but only via one of the
two subunits. The locations of the many other
GPI-anchored and transmembrane proteins
expressed by T. brucei are unknown.
In recent years, much attention has been
focused on the sorting of membrane proteins,
with the recognition that cellular membranes
are not lakes of randomly distributed lipids
but consist of lipid-specific subdomains. The
concept of lipid rafts has particular relevance
to GPI-anchored proteins, and suggests that
the very specific myristylation of the VSG GPI
anchor is probably an important determinant
of VSG secretion and recycling. T. brucei appears
to exert a high degree of quality control on VSG
secretion. Attempts to use bloodstream-form
T. brucei to express high levels of alien GPIanchored
proteins have failed. Even small modifications
of VSG structure are deleterious to
MOLECULAR BIOLOGY