Characterization of proteolytic degradation products of vaginally administered bovine lactoferrin

Abstract

When bovine lactoferrin (bLF) contacts human vaginal fluid (VF) it is subjected to proteolytic degradation. This report describes fragmentation patterns of bLF dosed vaginally in clinical trials or incubated ex vivo with VF. A consensus pattern of fragments was observed in samples from different women. The 80 kDa bLF molecule is initially cleaved between its homologous 40 kDa domains, the N-lobe and C-lobe, and then degraded into sub-fragments and mixtures of small peptides. We characterized this fragmentation process by polyacrylamide gel electrophoresis, western blotting, chromatographic separation, and mass spectral sequence analysis. Common to most VF fragmentation patterns were large amounts of an N-lobe 37 kDa fragment and a C-lobe 43 kDa fragment resulting from a single cleavage following tyrosine 324. Both fragments possessed full sets of iron-ligand amino acids and retained iron-binding ability. In some VF samples, alternative forms of large fragments were found, which like the 37+43 kDa pair, totaled 80 kDa. These included 58+22 kDa, 18+62 kDa, and 16+64 kDa forms. In general, the smaller component was from the N-lobe and the larger from the C-lobe. The 18+62 kDa pair was absent in some VF samples but highly abundant in others. This variability suggests multiple endopeptidases are involved, with the 18 kDa fragment’s presence dependent upon the balance of enzymes. Further action of VF endopeptidases produced smaller peptide fragments, and we found evidence that exopeptidases trimmed their N- and C-termini. The 3.1 kDa antimicrobial peptide lactoferricin B was not detected. These studies were facilitated by a novel technique we developed: tricolor western blots, which enabled simultaneous visualization of N- and C-terminal epitopes.

Antimicrobial activity of bovine lactoferrin against Gardnerella species clinical isolates

“…In addition, the antagonistic or synergistic effect of MTbLF and antimicrobials against resistant, intermediate, and sensitive presumptive G. vaginalis clinical isolates, were also investigated. The concentrations of iron, used in the present study, are in the range of iron levels measured in vaginal fluid (0.1–4 μg/ml; unpublished data) and concentrations of MTbLF used in cell culture are achieved and maintained in vaginal fluid following vaginal administration of MTbLF ( Hopp et al, 2022 ).…”

“…In addition, prolonged and repeated antibiotic treatments facilitate the development of resistant pathogens ( Beigi et al, 2004 ; Ferris et al, 2004 ; Bradshaw et al, 2006b ). In this context, resistance to both metronidazole and clindamycin is well documented among G. vaginalis isolates ( Knupp de Souza et al, 2016 ; Schuyler et al, 2016 ; Hopp et al, 2022 ), therefore complementary and/or alternative therapeutic approaches are needed.…”

Proteolysis of vaginally administered bovine lactoferrin: clearance, inter-subject variability, and implications for clinical dosing

“…While complete pharmacokinetic results are not yet available from that study, samples of vaginal fluid (VF) from a subset of patients enrolled and formulations tested have been used to characterize proteolysis and clearance of vaginally administered bLF. We recently reported the sequences of vaginal proteolysis products using a combination of western blotting, RP-HPLC fractionation, and peptide mass spectrometry (Hopp et al 2022 ). Herein, we extend those findings by examining the extent and variability of proteolysis as well as its overall contribution to clearance of vaginally administered bLF.…”

“…The predominant vaginal cleavage site for bLF is Tyr 324 yielding a 43 kDa C-lobe fragment and a 37 kDa N-lobe fragment, both of which retain intact iron-binding sequences (Hopp et al 2022 ). Other fragment pairs observed included 58 + 22 kDa, 18 + 62 kDa, and 16 + 64 kDa bands with the smaller component from the N-lobe and the larger from the C-lobe.…”

“…As we reported previously, it is important to assess fragmentation using both reducing and non-reducing gels, because bLF contains 17 disulfide bonds and some nicked species appear as less than 80 kDa on reducing gels but are likely still 80 kDa in vivo. Our previous report used a novel visualization method for bLF fragmentation, a tri-color western in which the results of western blotting with anti-bLF N-lobe and C-lobe monoclonal antibodies (mAbs) were shown in red and green, respectively, superimposed on PAGE gels of the same specimens stained with Coomassie blue (Hopp et al 2022 ). That same red, green, blue color convention is utilized in this report, where additional time-course PAGE gels are provided in order to follow the extent and variations of proteolysis.…”