Fig 1.
Fragmentation of bLF in human vaginal fluid.
Characteristic patterns of fragments developed when MTbLF was dosed intravaginally in an individual clinical subject and followed over time. On the left, samples from two time points, 12 and 24 hr post dose, are shown without reduction, preserving disulfide bonds. Identical samples are shown in the next two lanes, after reduction to break disulfide bonds revealing nicked fragments present in the 80 kDa forms on the left. At right, control samples are shown, including the subject’s VF with no bLF present and intact MTbLF before dosing, in their reduced forms. The numbers on the left and mid-right identify the molecular weights of fragments that we repeatedly detected in VF samples from many, but not all, clinical subjects dosed with MTbLF. The bLF control lane was loaded with 5 μg of MTbLF. Numbers at far right represent SeeBlue-Plus-2 standard proteins run on the same PAGE gels as the samples.
Fig 2.
Tricolor image of PAGE gel lanes overlaid with two westerns.
Selected time-point VF samples are shown for a second MTbLF-dosed clinical subject. To prepare this tricolor image, the three gels (Coomassie blue plus two mAb westerns) were put together in register by superimposing their SeeBlue standard ladders (weights shown at right), to match the gels precisely with each other. The molecular weight numbering scheme at middle right is colorized to indicate the major fragments along with their staining characteristics of red, green, or blue. Black numbers at 80 and 74 kDa indicate that those bands stained with all three stains. Green asterisks indicate the region where the red non-reduced bands disappear upon reduction and new, faint green bands appear (more on these green bands below). Blue numbers indicate bands that stained only with Coomassie blue, and hence lacked either epitope. Details of this new method of tricolor western imaging are given in the methods section and supplement S2 Fig.
Fig 3.
PAGE analysis of ex vivo VF digested MTbLF.
Coomassie blue stained PAGE gel. Bands present in the original MTbLF are labeled on the left. Fragment molecular weight numbers are shown in the same color scheme as in Fig 2. The standard protein labels at far right correspond to the SeeBlue-Plus-2 standard ladder in the center lane (MW). The fragment bands obtained in this experiment were mostly identical to bands in Fig 2.
Fig 4.
SEC HPLC of ex vivo digested MTbLF.
Both the 280 nm traces (above) and the 466 nm traces (below) are shown. Color codes show the time points of the digestion samples. The 280 nm profiles are superimposed while the 466 nm profiles are separated for easier comparisons. The two vertical gray bars indicate the elution positions of 80 kDa bLF (~15 min) and of the ~40 kDa peak (~16 min). 160 kDa bLF dimer elutes to the left of the main peak, under the time point numbers. The spiked peak to the left of dimer is the excluded volume of the column, where bLF trimer and higher polymers were expected to elute. The total volume of the column occurs at 22 minutes. Materials eluting later are present at T = 0 and therefore are likely to be VF peptides or other non-bLF substances which bind to the column packing material.
Fig 5.
RPC3 HPLC of MTbLF-dosed VF with non-reduced and reduced tricolor westerns.
The HPLC result in panel A shows a major peak at ~10 min for 80-kDa bLF and many earlier-eluting peaks corresponding to fragmented forms of bLF. In panels B and C, tricolor westerns of the HPLC fractions show many bands reactive with either the N-Lobe mAb (red) or the C-Lobe mAb (green), or non-epitope bearing but reactive with Coomassie stain (blue). Panel B shows samples electrophoresed without reduction, while Panel C shows the same samples after disulfide bonds were broken by reduction. Fragment molecular-weight numbers are shown, color-coded, at right. Asterisks indicate cases where separate peptides of the same molecular weight stain with different developing reagents in different lanes.
Fig 6.
Excision of fraction 19 PAGE bands for trypsinization and MS.
Fraction 19 of Fig 5C is shown with Coomassie blue staining only. Red boxes indicate bands excised from the gel, minced, destained, and trypsinized. Each trypsinized band was then subjected to LC-MS peptide mapping analysis.
Fig 7.
Plateau plots of peptide detection frequencies on MS.
Individual panels show results obtained for the trypsinized 43, 36, 30, 16, 15, and 13 kDa bands. The white-filled plateau traces represent all peptides with N- and C-termini matching expected tryptic cleavage sites. The gray-filled plateau traces show peptides with non-tryptic N- or C-termini. Cross-hatched bars indicate the four glycosylated tryptic peptides, which were not detected due to carbohydrate-moiety heterogeneity.
Fig 8.
Peptides with non-tryptic N-termini.
Two segments of the bLF sequence are shown, where peptides with non-tryptic N-termini were detected at high multiplicities. Panel A shows the region from sequence position 317 to 342 in which non-tryptic N-termini were found for the 43- and 30-kDa fragments, while Panel B shows the region from sequence position 563 to 585 in which non-tryptic N-termini were found for the 13-kDa fragment. Numbers above the sequences show the multiplicity of detection of peptides that start at those particular amino acids. These numbers correspond to the tallest gray-filled plateaus in the plots for these fragments in Fig 7. For clarity, positions with fewer than 5 detections are left blank.
Table 1.
Amino acid frequencies at N- and C-termini of fragments.
Fig 9.
Evidence for exopeptidase processing of fragments.
In panel A, a subset of fragments all starting at the N-terminus of bLF are aligned to show a series of C-terminal truncations consistent with processing of the longest fragment by carboxypeptidase(s) or other C-terminal exopeptidases. In panels B and C, fragments from the region 59–94 within bLF are arranged to show that both N-terminal truncations (B) and C-terminal truncations (C) can occur in the same set of peptides.
Fig 10.
Fragmentation model for bLF in vaginal fluid.
The central gray bar represents whole lactoferrin showing disulfide bonds (black lines), carbohydrate moieties (green asterisks), epitopes (yellow), and the antimicrobial peptides lactoferricin B (left) and lactoferrampin (middle) in red. The N- and/or C-termini of some fragments are approximate, their positions estimated only from PAGE and Western gels, which are only accurate to plus-or-minus 1–2 kDa. Others were defined precisely by mass spectroscopy as described in the text.
Fig 11.
Three-dimensional image of the largest fragments.
The 37 kDa N-lobe fragment is shown in red-and-yellow, and the 43 kDa C-lobe fragment is shown in green. The arrow near the center of the image shows the single peptide bond that was clipped to generate the two fragments. The iron atoms bound within each lobe are shown as orange spheres. The antimicrobial peptide portion of the N-lobe, lactoferricin B, is highlighted in yellow. It remains an integral portion of the 37 kDa fragment and reacts with its mAb on western blots. The C-lobe antigenic site is integral to its fragment as well. It is located on the far side of the green polypeptide segment and is not visible here.
Fig 12.
Sequence features of bLF and its VF fragments.
The 37 kDa N-lobe fragment is highlighted in pink and the 43 kDa C-lobe fragment in green. Various other structural features are mapped onto the sequence of bLF as noted in the legend: epitopes are highlighted in yellow, antimicrobial peptides are shown in red (lactoferricin B above, lactoferrampin below), carbohydrate-bearing asparagine (N) amino acids are noted with asterisks, basic amino acids targeted by limited tryptic digests are noted in purple [37] or blue [25]. The two identical homologous sets of iron-ligand amino acids are highlighted in orange; four occur in the 37 kDa fragment and four in the 43 kDa fragment.