Fig 1.
HAI-1 and HAI-2 complexes in human milk.
(A) Defatted human milk was loaded onto a CM-Sepharose column and the unbound fraction (UB) was collected. The milk (lanes 1) and the unbound fraction (lanes 2) were analyzed by immunoblot for HAI-1, HAI-2, and matriptase (MTP), as indicated. (B) Flow-chart summarizing the purification scheme for the HAI-1 and HAI-2 complexes in human milk. (C) Fractions 12 through 34 eluted from the CM-Sepharose chromatography were analyzed by Western blot for HAI-2. (D) The pooled HAI-2 containing fractions eluted from the CM-Sepharose were subjected to immunoaffinity chromatography on a HAI-1 mAb M19-Sepharose column followed by a HAI-2 mAb DC16-Sepharos column. Fractions 1 through 4 eluted from the mAb DC16-Sepharose were analyzed by Western blot for HAI-2 species (left panel). The pooled HAI-2 containing fractions were analyzed by immunoblot for matriptase species under non-boiled (lane 1) and boiled (lane 2) conditions (right panel).
Fig 2.
Purification and identification of prostasin-HAI-1 complex in human milk.
(A) The unbound fraction from the CM-Sepharose was further purified by DEAE-Sepharose chromatography. Fractions 10–21, eluted from the DEAE-Sepharose, were analyzed by Western blot for HAI-1 species. (B) The HAI-1 containing fractions eluted from the DEAE column were subjected to immunoaffinity chromatography using HAI-1 mAb M19-Sepharose. The eluted fraction was analyzed by SDS-PAGE for the protein profile (lane 1). MW stands for molecular weight markers. (C) The HAI-1 containing species from the immunoaffinity column were analyzed by 2-dimensional diagonal gel electrophoresis (gel panels on the left) and the indicated bands subjected to MS/MS-based protein identification (table on the right). First dimension electrophoresis was carried out under non-boiled conditions (upper panel); the second dimension was carried out after heat treating the gel strip sliced from the first dimension gel (lower panel). Six protein bands, as indicated, were sliced out for protein identification. The proteins, the number of peptides, and the relative abundance identified from the 6 bands are summarized in the table on the right.
Fig 3.
Purification and identification of HAI-2 complexes in human milk.
(A) The unbound fraction from the CM-Sepharose column was subjected to DEAE-Sepharose chromatography and the bound proteins eluted by a gradient of sodium chloride. The loaded fraction (lane L), flow-through fraction (FT), and eluted fractions 6–19, were analyzed by Western blot for HAI-2 species. (B) The DEAE fractions, containing HAI-2 species, were pooled and subjected to immunoaffinity chromatography using matriptase mAb 21-9-Sepharose followed by HAI-2 mAb DC16-Sepharose. The DEAE fraction (L), flow-through (FT), and two eluted fractions (E1 and E2) from the matriptase mAb-Sepharose were analyzed by Western blot (WB) for HAI-2 species (lanes 1–4) and matriptase (MTP) species (lanes 5–8). The eluted fractions (E1 and E2) from the HAI-2 mAb-Sepharose were analyzed by Western blot for HAI-2 species (lanes 9 and 10) and matriptase (MTP) species (lanes 11 and 12). (C) The HAI-2 species purified by immunoaffinity chromatography were analyzed by Western blot (WB) for HAI-2 species and by staining with Protoblue for the protein profile under non-boiled (N) and boiled (B) conditions. Seven protein bands, as indicated, were sliced from the gel for protein identification. The proteins, the number of peptides, and the relative abundance identified from the 7 gel bands are summarized in the table on the right.
Fig 4.
Characterization of prostasin monoclonal antibodies.
Three prostasin monoclonal antibodies were generated using purified prostasin-HAI-1 complex as the antigen. The proteins eluted from HAI-1 mAb M19-Sepharose (A.) or HAI-2 mAb DC16-Sepharose (B.) immunoaffinity columns were analyzed by immunoblot using the three prostasin mAbs, YL11, YL10, and YL89 under non-boiled, non-reducing conditions (lanes 1) or boiled, non-reducing conditions (lanes 2).
Fig 5.
Analysis of prostasin, matriptase, HAI-1 and HAI-2 species expressed by human mammary epithelial cells.
Lysates prepared from MTSV 1.7 milk-derived human mammary epithelial cells were analyzed by immunoblot for prostasin (Pros.), HAI-1, matriptase (MTP), and HAI-2 containing species before (lanes CL) and after (lanes HAI-1-D) immunodepletion of HAI-1 species from the lysates using HAI-1 mAb M19-Sepharose.
Fig 6.
Human mammary epithelial cells secrete prostasin and matriptase as complexes with the HAIs.
Conditioned medium collected from MTSV 1.7 milk-derived human mammary epithelial cells was analyzed by immunoblot for species containing matriptase (MTP), prostasin (Pros.), and HAI-1 before (lanes CM) and after (lanes HAI-1-D) immunodepletion of HAI-1 containing species with HAI-1 mAb M19-Sepharose. The conditioned medium was also analyzed by immunoblot for HAI-2 species after immunodepletion of prostasin (Pros.-D) or matriptase (MTP-D).
Fig 7.
A schematic illustration for putative roles of matriptase and prostasin in lactating mammary gland.