title: Human milk lactoferrin is a serine protease that cleaves Haemophilus surface proteins at arginine-rich sites.
abstract: Lactoferrin is a member of the lactotransferrin family of non-haem, iron-binding glycoproteins and is found at high concentrations in all human secretions, where it plays a major role in mucosal defence. In recent work, we observed that lactoferrin has proteolytic activity and attenuates the pathogenic potential of Haemophilus influenzae by cleaving and removing two putative colonization factors, namely the IgA1 protease protein and the Hap adhesin. Experiments with protease inhibitors further suggested that lactoferrin may belong to a serine protease family. In the present study we explored the mechanism of lactoferrin protease activity and discovered that mutation of either Ser259 or Lys73 results in a dramatic decrease in proteolysis. Examination of the crystal structure revealed that these two residues are located in the N-terminal lobe of the protein, adjacent to a 12-15 A cleft that separates the N-lobe and the C-lobe and that can readily accommodate large polypeptide substrates. In additional work, we found that lactoferrin cleaves IgA1 protease at an arginine-rich region defined by amino acids 1379-1386 (RRSRRSVR) and digests Hap at an arginine-rich sequence between amino acids 1016 and 1023 (VRSRRAAR). Based on our results, we conclude that lactoferrin is a serine protease capable of cleaving arginine-rich sequences. We speculate that Ser259 and Lys73 form a catalytic dyad, reminiscent of a number of bacterial serine proteases. In addition, we speculate that lactoferrin may cleave arginine-rich sequences in a variety of microbial virulence proteins, contributing to its long-recognized antimicrobial properties.
authors.affiliation: Edward Mallinckrodt Department of Pediatrics, Washington University School of Medicine, St Louis, MO 63110, USA.
authors.forename: D R
authors.initials: DR
authors.lastname: Hendrixson
authors.forename: J
authors.initials: J
authors.lastname: Qiu
authors.forename: S C
authors.initials: SC
authors.lastname: Shewry
authors.forename: D L
authors.initials: DL
authors.lastname: Fink
authors.forename: S
authors.initials: S
authors.lastname: Petty
authors.forename: E N
authors.initials: EN
authors.lastname: Baker
authors.forename: A G
authors.initials: AG
authors.lastname: Plaut
authors.forename: J W
authors.initials: JW
authors.lastname: St  Geme
mesh_terms: D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry; D001422:Bacterial Adhesion; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002460:Cell Line; D006193:Haemophilus influenzae / Q000187:drug effects / Q000201:enzymology*; D006801:Humans; D007781:Lactoferrin / Q000378:metabolism* / Q000494:pharmacology; D008895:Milk, Human / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D001425:Bacterial Outer Membrane Proteins; C091552:Hap protein, Hemophilus influenzae; D001120:Arginine; D007781:Lactoferrin; D012697:Serine Endopeptidases; C021433:IgA-specific serine endopeptidase