Direct evidence of the generation in human stomach of an antimicrobial peptide domain (lactoferricin) from ingested lactoferrin

The ability to define specific alterations in the structure and function of proteins as they are introduced and processed in vivo remains an important goal. We have evaluated the generation, in vivo, of an antimicrobial pepti de (lactoferricin) derived from ingested bovine lactoferrin by surface-enha nced laser desorption/ionization (SELDI). SELDI was used in the affinity ma ss spectrometry operational mode to detect and quantify lactoferricin direc tly from unfractionated gastric contents using a chemically defined ligand with a terminal n-butyl group as the lactoferricin affinity capture device. By this method, we were able to detect and quantify lactoferricin directly upon examination of unfractionated gastric contents recovered from an adul t subject 10 min after ingestion of bovine lactoferrin (200 mi of 10 mg/ml (1.2 x 10(-4) mol/l) solution). Lactoferricin produced in vivo was directly captured by a surface-enhanced affinity capture (SEAC) device composed of molecules with a terminal n-butyl group and analyzed by laser desorption/io nization time-of-flight mass spectrometry. The recovery of standard lactofe rricin or lactoferrin added to an aliquot of the gastric contents was deter mined to be nearly 100%, confirming the efficiency of this method. The amou nt of lactoferricin detected in the gastric contents was 16.9 +/- 2.7 mu g/ ml (5.4 +/- 0.8 x 10(-6) mol/l). However, a large proportion of ingested la ctoferrin was found to be incompletely hydrolyzed. Lactoferrin fragments co ntaining the lactoferricin region were analyzed by in situ pepsin hydrolysi s after being captured on the SEAC device. Partially degraded lactoferrin f ragments containing the lactoferricin region, including fragments correspon ding to positions 17-43, 17-44, 12-44, 9-58 and 16-79 of the bovine lactofe rrin sequence, were found to be present at concentrations as high as 5.7 +/ - 0.7 x 10(-5) mol/l. These results suggest that significant amounts of bov ine lactoferricin would be produced in the human stomach following ingestio n of food, such as infant formula, supplemented with bovine lactoferrin. We propose that physiologically functional quantities of human lactoferricin could be generated in the stomach of breast-fed infants, and possibly, in t he case of adults, from lactoferrin secreted into saliva. (C) 1998 Elsevier Science B.V. All rights reserved.