Binding of recombinant human proacrosin/acrosin to zona pellucida glycoproteins. II. Participation of mannose residues in the interaction.
OBJECTIVE
To assess the interaction of human proacrosin/acrosin with mannose residues coupled to a protein backbone.


METHODS
Prospective study.


METHODS
Basic research laboratory.


METHODS
Recombinant proteins derived from human proacrosin (Rec-40, Rec-30, Rec-20, Rec-10, and Rec-6) and bovine serum albumin (BSA)-mannose as ligand.


METHODS
In vitro binding assay developed to assess proacrosin/acrosin-BSA-mannose interaction.


METHODS
Proacrosin/acrosin binding to BSA-mannose; estimation of binding affinity.


RESULTS
All recombinant proteins of acrosin but Rec-6 (residues 1-59 of proacrosin) specifically bound to BSA-mannose. Rec-40 (proacrosin) showed the highest binding affinity (dissociation constant K(d), 162 nM), followed by N-terminal fragments Rec-30 (248 nM), Rec-20 (359 nM), and Rec-10 (402 nM). A significant decrease in binding activity was observed when acrosin proteins were treated with denaturing agents such as urea or heat. The beta-mercaptoethanol treatment produced a 39% decrease on Rec-30 binding to BSA-mannose; in contrast, no effect was observed with Rec-40, suggesting the presence of at least two types of mannose-binding sites.


CONCLUSIONS
[1] Proacrosin interacts with mannose residues through binding sites located at both the N- and C-terminal portion of the protein, [2] the full-length protein is required for maximal BSA-mannose binding, and [3] binding sites are stabilized by noncovalent bonds and by disulfide linkages.
Furlong
Laura I
LI
Instituto de Biología y Medicina Experimental-CONICET-UBA, Buenos Aires, Argentina.
Veaute
Carolina
C
Vazquez-Levin
Mónica H
MH
D000176:Acrosin / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D004527:Egg Proteins / Q000378:metabolism*; D004792:Enzyme Precursors / Q000378:metabolism*; D006801:Humans; D008297:Male; D008358:Mannose / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D011446:Prospective Studies; D011485:Protein Binding / Q000502:physiology; D025941:Protein Interaction Mapping; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism*; D000072216:Zona Pellucida Glycoproteins
D004527:Egg Proteins; D004792:Enzyme Precursors; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C082833:ZP4 protein, human; D000072216:Zona Pellucida Glycoproteins; C022438:proacrosin; D000176:Acrosin; D008358:Mannose