Origin and Mechanism of Evolution of Antifreeze Glycoproteins in Polar Fishes
315
AFGP gene is shown in Fig. lA. It consists of two exons interrupted by a
single large intron (II, about 1.9 kbp). The small exon 1 (El) encodes the
5'UTR (untranslated region) and the signal peptide, and the large exon 2
(E2) encodes the AFGPs and 3'UTR. Other notothenioid AFGP genes have
the same structures (unpublished data).
Notothenioid AFGP genes have an unusual polyprotein structure in the
AFGP coding region (E2) (Fig. lA). While most protein genes encode one
protein molecule per gene, each notothenioid AFGP gene encodes a
polyprotein precursor containing many AFGP molecules, as many as 46 in
the Nc (Notothenia coriiceps) gene [32]. These molecules are linked in
tandem by 3-residue spacers with the highly conserved sequence of LIFI/N-F (LeulPhe-Ile/Asn-Phe) (Fig. lB) [12,29,30,32]. Since the spacer
residues are not found in the mature AFGPs, and Leu and Phe are known
substrates of chymotrypsin, it is very likely that the AFGP gene is
transcribed and translated into a polyprotein which is then cleaved posttranslationally at the spacers by a chymotrypsin-like protease to yield the
individual AFGP molecules (Fig. lB).
The one Nc and two Dm AFGP genes that we have reported all encode
the small AFGP isoforms, AFGPs 6, 7 and 8, encompassing their various
Pro (substituting Ala) variants [29,30,32]. We have now also obtained
another gene from Dm that encodes two large size isoforms, in the range of
AFGP4 and AFGPI respectively, among five other small ones (manuscript
in preparation). These gene sequence data provided two pieces of
important information regarding AFGP heterogeneity and abundance.
First, each and every size and compositional AFGP isoform is distinctly
encoded as individual copies within polyprotein genes. Thus the various
lengths are not due to protein processing through splicing small AFGPs or
cleaving large AFGPs into small ones. Second, a large family of
polyprotein genes [12] can rapidly escalate the gene dosage that must
contribute to the circulatory abundance (35 mg/ml) of the AFGP in these
fishes.
Notothenioid AFGP Gene Evolved from a Pancreatic
Trypsinogen Gene
The first clue to the origin of notothenioid AFGP gene was obtained from
a data base search for similar sequences, using the three AFGP genes that
encode small AFGPs as queries. The 3' flanking sequences of these AFGP
genes were found to share over 70% identity with the 3' end of a
trypsinogen cDNA from a pleuronectiform teleost, Atlantic plaice,
Pleuronectes platessa [33]. This high degree of nucleotide sequence
identity clearly signals that notothenioid AFGP and trypsinogen genes are
315
AFGP gene is shown in Fig. lA. It consists of two exons interrupted by a
single large intron (II, about 1.9 kbp). The small exon 1 (El) encodes the
5'UTR (untranslated region) and the signal peptide, and the large exon 2
(E2) encodes the AFGPs and 3'UTR. Other notothenioid AFGP genes have
the same structures (unpublished data).
Notothenioid AFGP genes have an unusual polyprotein structure in the
AFGP coding region (E2) (Fig. lA). While most protein genes encode one
protein molecule per gene, each notothenioid AFGP gene encodes a
polyprotein precursor containing many AFGP molecules, as many as 46 in
the Nc (Notothenia coriiceps) gene [32]. These molecules are linked in
tandem by 3-residue spacers with the highly conserved sequence of LIFI/N-F (LeulPhe-Ile/Asn-Phe) (Fig. lB) [12,29,30,32]. Since the spacer
residues are not found in the mature AFGPs, and Leu and Phe are known
substrates of chymotrypsin, it is very likely that the AFGP gene is
transcribed and translated into a polyprotein which is then cleaved posttranslationally at the spacers by a chymotrypsin-like protease to yield the
individual AFGP molecules (Fig. lB).
The one Nc and two Dm AFGP genes that we have reported all encode
the small AFGP isoforms, AFGPs 6, 7 and 8, encompassing their various
Pro (substituting Ala) variants [29,30,32]. We have now also obtained
another gene from Dm that encodes two large size isoforms, in the range of
AFGP4 and AFGPI respectively, among five other small ones (manuscript
in preparation). These gene sequence data provided two pieces of
important information regarding AFGP heterogeneity and abundance.
First, each and every size and compositional AFGP isoform is distinctly
encoded as individual copies within polyprotein genes. Thus the various
lengths are not due to protein processing through splicing small AFGPs or
cleaving large AFGPs into small ones. Second, a large family of
polyprotein genes [12] can rapidly escalate the gene dosage that must
contribute to the circulatory abundance (35 mg/ml) of the AFGP in these
fishes.
Notothenioid AFGP Gene Evolved from a Pancreatic
Trypsinogen Gene
The first clue to the origin of notothenioid AFGP gene was obtained from
a data base search for similar sequences, using the three AFGP genes that
encode small AFGPs as queries. The 3' flanking sequences of these AFGP
genes were found to share over 70% identity with the 3' end of a
trypsinogen cDNA from a pleuronectiform teleost, Atlantic plaice,
Pleuronectes platessa [33]. This high degree of nucleotide sequence
identity clearly signals that notothenioid AFGP and trypsinogen genes are