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Cysteine-Rich Atrial Secretory Protein from the Snail Achatina achatina: Purification and Structural Characterization.

Shabelnikov S, Kiselev A - PLoS ONE (2015)

Bottom Line: Despite extensive studies of cardiac bioactive peptides and their functions in molluscs, soluble proteins expressed in the heart and secreted into the circulation have not yet been reported.A combination of fold recognition algorithms and ab initio folding predicted that CRASP adopts an all-alpha, right-handed superhelical fold.CRASP is the first secretory protein expressed in molluscan atrium to be reported.

View Article: PubMed Central - PubMed

Affiliation: Department of Cytology and Histology, Saint-Petersburg State University, St. Petersburg, Russia; Laboratory of Cell Morphology, Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russia.

ABSTRACT
Despite extensive studies of cardiac bioactive peptides and their functions in molluscs, soluble proteins expressed in the heart and secreted into the circulation have not yet been reported. In this study, we describe an 18.1-kDa, cysteine-rich atrial secretory protein (CRASP) isolated from the terrestrial snail Achatina achatina that has no detectable sequence similarity to any known protein or nucleotide sequence. CRASP is an acidic, 158-residue, N-glycosylated protein composed of eight alpha-helical segments stabilized with five disulphide bonds. A combination of fold recognition algorithms and ab initio folding predicted that CRASP adopts an all-alpha, right-handed superhelical fold. CRASP is most strongly expressed in the atrium in secretory atrial granular cells, and substantial amounts of CRASP are released from the heart upon nerve stimulation. CRASP is detected in the haemolymph of intact animals at nanomolar concentrations. CRASP is the first secretory protein expressed in molluscan atrium to be reported. We propose that CRASP is an example of a taxonomically restricted gene that might be responsible for adaptations specific for terrestrial pulmonates.

No MeSH data available.


Related in: MedlinePlus

Purification of CRASP isoforms from the atria of A. achatina.(A) Semi-preparative size-exclusion chromatography on a Superdex 200 column. Fraction from 31 to 34 min (shaded grey) was collected and subjected to anion exchange chromatography. (B) Separation of isoforms on a Mono-Q anion exchange column. Fractions from the two major peaks were designated CRASP-A and CRASP-B. Final purification of CRASP-A (C) and CRASP-B (D) on a ProRPC C4 reversed phase column. (E) All fractions were analysed with SDS-PAGE (15% gel) and silver staining.
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pone.0138787.g001: Purification of CRASP isoforms from the atria of A. achatina.(A) Semi-preparative size-exclusion chromatography on a Superdex 200 column. Fraction from 31 to 34 min (shaded grey) was collected and subjected to anion exchange chromatography. (B) Separation of isoforms on a Mono-Q anion exchange column. Fractions from the two major peaks were designated CRASP-A and CRASP-B. Final purification of CRASP-A (C) and CRASP-B (D) on a ProRPC C4 reversed phase column. (E) All fractions were analysed with SDS-PAGE (15% gel) and silver staining.

Mentions: CRASP was isolated from the atria of A. achatina snails through a combination of size-exclusion, anion exchange and reversed phase chromatography (Fig 1). In the first purification step, CRASP was obtained in a single peak with a retention time of about 32 min (Fig 1A). Subsequent anion exchange purification yielded two peaks with the same mobility on SDS-PAGE (Fig 1B and 1E), indicating the presence of two distinct isoforms. Pooled fractions from these peaks, were designated CRASP-A and CRASP-B, eluted at ~136 mM and ~160 mM NaCl, respectively. The native proteins fractions were used for analytical isoelectric focusing and structural studies in size-exclusion chromatography and CD spectroscopy. After a final reversed phase HPLC purification, we obtained virtually homogeneous samples of CRASP-A (Fig 1C and 1E) and CRASP-B (Fig 1D and 1E), which were used for Edman degradation and mass spectrometry.


Cysteine-Rich Atrial Secretory Protein from the Snail Achatina achatina: Purification and Structural Characterization.

Shabelnikov S, Kiselev A - PLoS ONE (2015)

Purification of CRASP isoforms from the atria of A. achatina.(A) Semi-preparative size-exclusion chromatography on a Superdex 200 column. Fraction from 31 to 34 min (shaded grey) was collected and subjected to anion exchange chromatography. (B) Separation of isoforms on a Mono-Q anion exchange column. Fractions from the two major peaks were designated CRASP-A and CRASP-B. Final purification of CRASP-A (C) and CRASP-B (D) on a ProRPC C4 reversed phase column. (E) All fractions were analysed with SDS-PAGE (15% gel) and silver staining.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4596865&req=5

pone.0138787.g001: Purification of CRASP isoforms from the atria of A. achatina.(A) Semi-preparative size-exclusion chromatography on a Superdex 200 column. Fraction from 31 to 34 min (shaded grey) was collected and subjected to anion exchange chromatography. (B) Separation of isoforms on a Mono-Q anion exchange column. Fractions from the two major peaks were designated CRASP-A and CRASP-B. Final purification of CRASP-A (C) and CRASP-B (D) on a ProRPC C4 reversed phase column. (E) All fractions were analysed with SDS-PAGE (15% gel) and silver staining.
Mentions: CRASP was isolated from the atria of A. achatina snails through a combination of size-exclusion, anion exchange and reversed phase chromatography (Fig 1). In the first purification step, CRASP was obtained in a single peak with a retention time of about 32 min (Fig 1A). Subsequent anion exchange purification yielded two peaks with the same mobility on SDS-PAGE (Fig 1B and 1E), indicating the presence of two distinct isoforms. Pooled fractions from these peaks, were designated CRASP-A and CRASP-B, eluted at ~136 mM and ~160 mM NaCl, respectively. The native proteins fractions were used for analytical isoelectric focusing and structural studies in size-exclusion chromatography and CD spectroscopy. After a final reversed phase HPLC purification, we obtained virtually homogeneous samples of CRASP-A (Fig 1C and 1E) and CRASP-B (Fig 1D and 1E), which were used for Edman degradation and mass spectrometry.

Bottom Line: Despite extensive studies of cardiac bioactive peptides and their functions in molluscs, soluble proteins expressed in the heart and secreted into the circulation have not yet been reported.A combination of fold recognition algorithms and ab initio folding predicted that CRASP adopts an all-alpha, right-handed superhelical fold.CRASP is the first secretory protein expressed in molluscan atrium to be reported.

View Article: PubMed Central - PubMed

Affiliation: Department of Cytology and Histology, Saint-Petersburg State University, St. Petersburg, Russia; Laboratory of Cell Morphology, Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russia.

ABSTRACT
Despite extensive studies of cardiac bioactive peptides and their functions in molluscs, soluble proteins expressed in the heart and secreted into the circulation have not yet been reported. In this study, we describe an 18.1-kDa, cysteine-rich atrial secretory protein (CRASP) isolated from the terrestrial snail Achatina achatina that has no detectable sequence similarity to any known protein or nucleotide sequence. CRASP is an acidic, 158-residue, N-glycosylated protein composed of eight alpha-helical segments stabilized with five disulphide bonds. A combination of fold recognition algorithms and ab initio folding predicted that CRASP adopts an all-alpha, right-handed superhelical fold. CRASP is most strongly expressed in the atrium in secretory atrial granular cells, and substantial amounts of CRASP are released from the heart upon nerve stimulation. CRASP is detected in the haemolymph of intact animals at nanomolar concentrations. CRASP is the first secretory protein expressed in molluscan atrium to be reported. We propose that CRASP is an example of a taxonomically restricted gene that might be responsible for adaptations specific for terrestrial pulmonates.

No MeSH data available.


Related in: MedlinePlus