Biomedical subjects

S Y Gauthier

Publications and source records attributed to S Y Gauthier.

6 recordsLinked to original sources

PubMed1999

Structure and expression of the highly repetitive histone H1-related sperm chromatin proteins from winter flounder.

In the late stages of spermatogenesis, winter flounder produce a family of high molecular mass (80-200 kDa) basic nuclear proteins (HMrBNPs) that combine with the normal complement of histones to produce condensed sperm chromatin with an increased nucleosomal repeat length. The HMrBNPs have a biased amino-acid composition in which Arg, Ser, Lys and Pro are abundant because of their presence in many simple peptide repeats. The organization of these repeats was deduced by cDNA cloning. The predominant repeating units are related 26- and 30-amino-acid sequences that in turn are linked by 6-amino-acid spacers to form 58- and 62-amino-acid repeats. Subsets of these repeats are also present, such as a dispersed 20-amino-acid repeat and a tandem array of nine heptapeptides at the C-terminus. The HMrBNPs appear to have evolved from an extreme H1 variant that has an N-terminal tail of HMrBNP-like sequence linked to an H1 globular region. Based on sequences of the most abundant HMrBNP cDNAs, and the lack of hybridization between HMrBNP mRNAs and a DNA probe for the H1 globular region, the latter domain appears to have been lost during expansion and amplification of the HMrBNP-like repeats. Transcripts of the HMrBNP and H1 variant genes are present in testis RNAs only during the mid-spermatid stage of spermatogenesis, at the same time that HMrBNPs in their highly phosphorylated form first appear in the nucleus. Judging by the lack of a lag between HMrBNP mRNA synthesis and translation, the mRNAs for these highly basic proteins are not stored for any length of time. Instead, the deposition of HMrBNPs onto DNA, which coincides with the major reorganization and silencing of the chromatin, may be controlled by dephosphorylation.

Amino Acid Sequence↗

PubMed1998

Disulfide bond mapping and structural characterization of spruce budworm antifreeze protein.

The 9-kDa, Thr-, Ser-, and Cys-rich thermal hysteresis protein from spruce budworm (sbwTHP) is 10-30 times more effective than fish antifreeze proteins (AFPs) at depressing solution freezing points via ice-crystal growth inhibition. Since this insect protein is only available in microgram quantities from its natural source, recombinant sbwTHP was produced from inclusion bodies in Escherichia coli by a refolding protocol. Incompletely folded forms were removed during ion-exchange and reverse-phase chromatography, resulting in fully active sbwTHP that was indistinguishable in its properties from native sbwTHP. The antifreeze was completely inactivated by reduction, showed no reaction with sulfhydryl reagents, and was not inhibited by EDTA. All eight cysteine residues appear to be involved in disulfide bond formation. Tryptic cleavage and peptide analysis is consistent with linkages between the first and second cysteine residues, the third and fourth, fifth and eighth, and the sixth and seventh. NMR analysis confirmed that the fully folded form of sbwTHP was well structured and had a single conformation. Both NMR and CD spectra indicate the presence of extensive beta structure (70-80%) with little or no alpha helix. The protein maintains antifreeze activity over a broad range of pH values, and its conformation is independent of both temperature (over the range 0 degrees C to 20 degrees C), and the presence of 50% trifluoroethanol.

Amino Acid Sequence↗

PubMed1996

Evidence for a proprotein intermediate during maturation of type II antifreeze protein in sea raven, Hemitripterus americanus.

The circulating Type II antifreeze protein (AFP) in sea raven is 129 amino acids (aa) long (14 kDa) and is derived from an initial 163 aa translation product that is synthesised in the liver. Signal peptide cleavage algorithms, as well as transgenic expression studies in fall armyworm cells, predict the formation of a 146 aa (16 kDa) proprotein intermediate. A protein of this size that cross-reacted with anti-sea raven AFP antibody was detected in sea raven serum using phosphate/urea SDS-PAGE, and was purified by size-exclusion chromatography and reverse-phase HPLC. N-terminal sequencing and mass spectrometry identified the protein as the predicted proAFP, and immunoblotting suggested that it is the predominant form present in liver. These results are consistent with production and storage of a proAFP intermediate in the liver, and its subsequent processing to mature AFP during or soon after its release into the circulation.

Animals↗

PubMed1996

A natural variant of type I antifreeze protein with four ice-binding repeats is a particularly potent antifreeze.

A 4.3-kDa variant of Type I antifreeze protein (AFP9) was purified from winter flounder serum by size exclusion chromatography and reversed-phase HPLC. By the criteria of mass, amino acid composition, and N-terminal sequences of tryptic peptides, this variant is the posttranslationally modified product of the previously characterized AFP gene 21a. It has 52 amino acids and contains four 11-amino acid repeats, one more than the major serum AFP components. The larger protein is completely alpha-helical at 0 degree C, with a melting temperature of 18 degrees C. It is considerably more active as an antifreeze than the three-repeat winter flounder AFP and the four-repeat yellowtail flounder AFP, both on a molar and a mg/mL basis. Several structural features of the four-repeat winter flounder AFP, including its larger size, additional ice-binding residues, and differences in ice-binding motifs might contribute to its greater activity. Its abundance in flounder serum, together with its potency as an antifreeze, suggest that AFP9 makes a significant contribution to the overall freezing point depression of the host.

Amino Acid Sequence↗

PubMed1994

Cystine-rich fish antifreeze is produced as an active proprotein precursor in fall armyworm cells.

Recombinant cystine-rich fish antifreeze protein (AFP) was produced by fall armyworm cells from a baculovirus expression vector containing sea raven AFP cDNA. The natural signal sequence encoded in the cDNA directed secretion of the antifreeze into the medium, from where it was recovered and purified to homogeneity. The M(r) of the exported protein (16k), as determined by SDS-PAGE, was larger than that (14k) of mature AFP isolated from sea raven serum. Sequencing of the recombinant AFP showed that it had a 17-amino-acid extension N-terminal to the 129-amino-acid mature AFP that began where signal peptide cleavage should occur according to current algorithms. Recombinant proAFP had antifreeze activity equivalent to that of the mature AFP, which indicates that the disulfide bonds were correctly formed and that the ice-binding site on the antifreeze is not sterically hindered by the 17-amino-acid N-terminal extension.

Amino Acid Sequence↗

PubMed1992

Antifreeze protein pseudogenes.

Three members, 11-3, F2 and 5a, of the type-I antifreeze protein (AFP) multigene family in winter flounder were sequenced. All three belong to the subset of AFP genes that are linked, but irregularly spaced, and show significant differences from the functional genes in tandem repeats. 11-3 and F2 appear to be pseudogenes. Their intron, 3'-exon and 3'-flanking DNAs are similar to those of other AFP genes, but their 5'-exon is either missing or extensively modified, and has stop codons present in all three reading frames. Based on a comparison of intron sequences of family members, 11-3/F2 may represent a residual progenitor AFP gene which was duplicated after reaching pseudogene status. The third gene, 5a, is remarkable in having a 3'-exon that encodes an exceptionally long, Ala-rich sequence that lacks any semblance of the 11-amino acid repeats found in 11-3, F2 and functional AFP genes. 5a might also be a pseudogene, because its presumed TATA box appears to have mutated.

Amino Acid Sequence↗