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

• Published in: Biochimica et biophysica acta Vol. 1429; no. 1; pp. 129 - 141
• Main Authors: Kuwata, Hidefumi, Yip, Tai-Tung, Tomita, Mamoru, Hutchens, T.William
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 08-12-1998
• Subjects: Amino Acid Sequence; Binding Sites; Digestion; Gastric Mucosa - metabolism; Gastrointestinal Contents - chemistry; Gastrointestinal Contents - microbiology; Humans; Ingestion; Intestines - microbiology; Lactoferricin; Lactoferrin; Lactoferrin - administration & dosage; Lactoferrin - analogs & derivatives; Lactoferrin - biosynthesis; Lactoferrin - chemistry; Lactoferrin - metabolism; Male; Mass spectrometry; Peptide Fragments - analysis; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods; Surface-enhanced laser desorption/ionization; Time Factors; Lactoferricin; BSA, bovine serum albumin; PMSF, phenylmethanesulfonyl fluoride; SELDI, surface-enhanced laser desorption/ionization; Digestion; HRG, histidine rich glycoprotein; Surface-enhanced laser desorption/ionization; ELISA, enzyme-linked immunosorbent assay; SEAC, surface-enhanced affinity capture; HPLC, high performance liquid chromatography; Ingestion; Lfcin, lactoferricin; MALDI, matrix-assisted laser desorption/ionization; Lactoferrin; PBS, phosphate-buffered saline; bLfcin, bovine lactoferricin; LPS, lipopolysaccharide; Mass spectrometry; hLfcin, human lactoferricin
• ISSN: 0167-4838; 0006-3002; 1879-2588
• DOI: 10.1016/S0167-4838(98)00224-6

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 peptide (lactoferricin) derived from ingested bovine lactoferrin by surface-enhanced laser desorption/ionization (SELDI). SELDI was used in the affinity mass spectrometry operational mode to detect and quantify lactoferricin directly 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 adult subject 10 min after ingestion of bovine lactoferrin (200 ml of 10 mg/ml (1.2×10
−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/ionization time-of-flight mass spectrometry. The recovery of standard lactoferricin or lactoferrin added to an aliquot of the gastric contents was determined to be nearly 100%, confirming the efficiency of this method. The amount of lactoferricin detected in the gastric contents was 16.9±2.7 μg/ml (5.4±0.8×10
−6 mol/l). However, a large proportion of ingested lactoferrin was found to be incompletely hydrolyzed. Lactoferrin fragments containing the lactoferricin region were analyzed by in situ pepsin hydrolysis after being captured on the SEAC device. Partially degraded lactoferrin fragments containing the lactoferricin region, including fragments corresponding to positions 17–43, 17–44, 12–44, 9–58 and 16–79 of the bovine lactoferrin sequence, were found to be present at concentrations as high as 5.7±0.7×10
−5 mol/l. These results suggest that significant amounts of bovine lactoferricin would be produced in the human stomach following ingestion 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 the case of adults, from lactoferrin secreted into saliva.