Purificação, caracterização bioquímica e estrutural de duas novas lectinas da esponja marinha Aplysina Pseudolacunosa (Pinheiro; Hajdu, 2007)

Abstract

Marine sponges, belonging to the phylum Porifera, are important sources of bioactive biomolecules, including lectins, which are proteins capable of recognizing and reversibly binding to specific carbohydrates. The species Aplysina pseudolacunosa, found along the coast of Ceará, had not yet had any lectins characterized in the literature. Therefore, this study aimed to purify and biochemically and structurally characterize two lectins from this species, named ApL-1 and ApL-2. The lectins were purified by ammonium sulfate precipitation, followed by affinity chromatography and reverse-phase high-performance liquid chromatography. In SDS- PAGE, ApL-1 exhibited an apparent molecular mass of approximately 12 kDa, whereas ApL-2 showed a mass of about 28 kDa. Both lectins displayed hemagglutinating activity, with titers of 32 U.H. mL−1 for ApL-1 and 128 U.H. mL−1 for ApL-2. ApL-1 showed greater affinity to bovine fetuin, while ApL-2 was strongly inhibited by porcine gastric mucins types II and III. Both lectins maintained activity over a wide pH range (4–10), exhibited thermal stability and divalent ion–independent activity. Circular dichroism characterization revealed distinct conformational architectures, with a predominance of β-sheet structures for ApL-1, consistent with galectins described from marine sponges, whereas ApL-2 exhibited a structural organization with a higher content of α-helical structures. Dynamic light scattering analyses indicated a monodisperse behavior for ApL-1, suggesting a dimeric organization in solution, and a more heterogeneous profile for ApL-2, indicating a higher degree of molecular association. The primary structure of ApL-1 was fully elucidated by tandem mass spectrometry (MS/MS). BLASTp analyses revealed high homology with galectins from marine sponges, particularly the isoforms ALL-a and ALL-b, with sequence identities of 86.9% and 78.6%, respectively, and 100% sequence coverage, compatible with the structural pattern described for proto-galectins. ApL-2 had its primary structure partially characterized, and alignments with putative proteins from marine sponge genomes showed identities ranging from 34 to 41% with low sequence coverage, suggesting that it represents a non-conventional lectin. Thus, the two new lectins characterized in this study expand the current knowledge of structural and functional diversity among marine sponge lectins and provide a foundation for future biological characterization and potential biotechnological investigation, pending further functional validation.