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NEMATODES 315
Most tissue in the underlying layers of the
cuticle consists of collagen or collagen-like
proteins. Collagen is the most abundant protein
in nematodes, as in other metazoa, comprising
about 1% of the mass of adults. In
C. elegans, two types of collagen have been
identified. These correspond to two types of
extracellular matrix, the cuticle and basement
membranes, including a layer that surrounds
the hypodermis. Collagens are characterized
by repeats of the sequence X-Y-Gly, where X is
usually L-proline and Y is 4-hydroxy-L-proline.
Polypeptide chains containing these repeats
are wound into tight triple helices. The
hydroxylation of proline residues is catalyzed
by prolyl 4-hydroxylase, a tetramer composed
of 2 and 2 subunits. In C. elegans, selective
deletion of dpy-18, which encodes the -subunit,
results in mutants with shorter (or
‘dumpy’) body shapes. In contrast, deletion of
phy-2, which encodes the -subunit, does not
result in a phenotypic change. However, crossing
phy-2 with dpy-18 worms yields progeny
that are unable to synthesize cuticle
collagens, including those that form the basement
membrane of the hypodermis. In addition
to expressing the dumpy phenotype, a
high percentage of these worms explode following
gradual structural breakdown or
breaches of the cuticle. These mutations illustrate
the crucial role of a resistive structure at
the external surface of nematodes.
Nematode cuticular collagens contain
higher levels of proline (11%) and hydroxyproline
(12%) than vertebrate collagens. About
80% of the total cuticular protein in nematodes
(C. elegans, A. suum and Haemonchus
contortus) consists of 2-mercaptoethanolsoluble
collagen. Most of the remaining 20%
consists of highly insoluble proteins, such as a
keratin-like protein and cuticulin, a protein
that is highly crosslinked through tyrosine
residues. Tyrosine crosslinkages are achieved
by peroxidation, leading to dityrosine residues,
but also include an unusual isotrityrosine
component, the synthesis of which remains
unresolved. These insoluble proteins are
located primarily in the cortex. Structural
analyses of ASCUT-1, a cuticulin found in
Ascaris lumbricoides, demonstrate the critical
nature of phenylalanine residues to intramolecular
interactions between cuticulin
molecules, which are essential for aggregation
and cuticle formation.
Most cuticle protein assembly occurs in the
hypodermis, and mRNAs for several genes
involved in cuticle synthesis have been localized
in the hypodermis of C. elegans. These
include an amino acid hydroxylase that converts
phenylalanine to tyrosine, which is critical
to the process of protein crosslinking, and
two cuticulins that localize to the external
cortical layer. About 150 distinct collagen
genes have been identified in C. elegans, and
homologs are beginning to emerge from EST
projects for parasitic species. Gene products
found in cuticle are typically short (300 amino
acids) and fall into four families. Cuticle collagen
genes in parasitic species, including
A. suum and H. contortus, as well as the filariid,
B. pahangi, also group into evolutionarily conserved
families. However, most information
about the genetics of nematode cuticle collagens
has been derived from analyses of C. elegans.
Mutations in collagen genes cause specific
morphological (and sometimes behavioral)
abnormalities that reflect the localization and
function of the gene product. At least 50 genes
in C. elegans affect body morphology, and eight
of these encode collagens. For example, sqt-1
encodes a cuticle collagen that is important in
determining shape and locomotory behavior.
Some mutations in sqt-1 produce left-handed
helical twisting of the nematode, which results
in worms (left-rollers) that move only in circular
paths. Careful observation of the structure of the
BIOCHEMISTRY AND CELL BIOLOGY: HELMINTHS