trans

228 TRYPANOSOMATID CARBOHYDRATES
Interestingly, when the parasites are grown in
culture, the -galactose termini of these side
chains are terminated with sialic acid by the
action of a trans-sialidase enzyme. Recent
work has shown that GPEET-procyclin is
phosphorylated.
The various procyclins appear to be expressed
in a cell cycle-specific manner. Early in
infection GPEET2 is the only procyclin detected.
However, GPEET2 disappears in about a week
and is replaced by several isoforms of glycosylated
EP, but not the unglycosylated isoform
EP2. Unexpectedly, the N-terminal domains
of all procyclins appear to be quantitatively
removed by proteolysis in the fly, but not
in culture. These findings suggest that one
function of the protease-resistant C-terminal
domain, containing the amino acid repeats, is
to protect the parasite surface from digestive
enzymes in the tsetse fly gut. Moreover, it has
been hypothesized that the oligosaccharide
chains may serve as lectin-binding ligands
within the tsetse fly midgut. Based on their
polyanionic nature and extended conformation,
a likely function of procyclins is to afford
protection of the parasite in the digestive
confines of the tsetse fly midgut.
Intriguingly, while compelling evidence has
been obtained that GPI biosynthesis is essential
to the bloodstream forms of T. brucei, GPI
biosynthesis in procyclic forms is non-essential,
even though procyclics are rich in the GPIanchored
procyclins. Upon deletion of individual
genes of the procyclin family, all but one
could be deleted before producing non-viable
parasites. To study the function of procyclins,
mutants have been generated in the Roditi
laboratory that have no EP genes and only one
copy of GPEET. This last gene could not be
replaced by EP procyclins, and could only be
deleted once a second GPEET copy was introduced
into another locus. The EP knockouts
are morphologically indistinguishable from
the parental strain, but their ability to establish
a heavy infection in the insect midgut is
severely compromised; this phenotype can
be reversed by the reintroduction of a single,
highly expressed EP gene. These results suggest
that the two types of procyclin have different
roles, and that the EP form, while not
required in culture, is important for survival in
the fly.
Mucins
TRYPANOSOMA CRUZI
T. cruzi has a dense and continuous coat composed
of a layer of type-I glycosylinositolphospholipids
or GIPLs, and a family of small
mucins that project above the GIPL layer. The
GIPLs have the same anchor structure as the
T. brucei procyclins but are heavily substituted
with Gal, GlcNAc and sialic acid. The mucins
are rich in threonine, serine and proline
residues that are heavily glycosylated with
O-linked GlcNAc as the internal residue, and
further modified with 1–5 galactosyl residues.
Two groups of mucin-like molecules have been
identified and are encoded by the diverse MUC
gene family. The glycoproteins in the first
group are 35–50 kDa in size and are expressed
on the parasitic forms found in the insect
(epimastigote and metacyclic trypomastigote).
The second group consists of 80–200 kDa
glycoproteins that are expressed in the cell
culture-derived trypomastigotes. These glycoconjugates
express the Ssp-3 epitope, which
were later called F2/3 glycoproteins.
The glycan structures vary with the developmental
stages of the parasite, a notable modification
being the trans-sialylation of the glycan
in the metacyclic stages. Trypanosomes are
unable to synthesize sialic acid de novo, but
do have the unusual ability to acquire it from
BIOCHEMISTRY AND CELL BIOLOGY: PROTOZOA