Characterization of receptors for murine pregnancy specific glycoproteins 17 and 23

Abstract

In primates and rodents, trophoblast cells synthesize and secrete into the maternal circulation a family of proteins known as pregnancy specific glycoproteins (PSG). The current study was undertaken to characterize the receptor for two members of the murine PSG family, PSG17 and PSG23. Binding of recombinant PSG17 and PSG23 to CHO-K1 and L929 cells and their derived mutants was performed to determine whether these proteins bound to cell surface proteoglycans. We also examined binding of these proteins to cells transfected with syndecans and glypican-1 by flow cytometry. The interaction with glycosaminoglycans was confirmed in solid phase assays. Our results show that PSG17 binds to CD9 and to cell surface proteoglycans while PSG23 binds only to the latter. We found that the amino acids involved in CD9 binding reside in the region of highest divergence between the N1-domains of murine PSGs. For both proteins, the N-terminal domain (designated as N1) is sufficient for binding to cells and the ability to bind cell surface proteoglycans is affected by the cell line employed to generate the recombinant proteins. We conclude that while substantially different at the amino acid level, some murine PSGs share with human PSG1 the ability to bind to cell surface proteoglycans and that at least one PSG binds to more than one type of molecule on the cell surface.

Figure 1

Protein characterization, binding of PSGs to different cell lines, and analysis of CD9 expression in L929 cells. (A) Coomassie stained SDS-PAGE gel showing the recombinant proteins after purification. Lane 1: FLAG-Fc, lane 2: PSG17N-Fc, and lane 3: PSG23N-Fc. The molecular weight marker on the left indicates the sizes in kilodaltons. L929, CHO-K1, Sog9 or CHO-pgsA-745 cells were incubated with PSG23N-Fc or PSG23N1AHisFLAG or FLAG-Fc as indicated (B) or with the control protein and PSG17N-Fc (D) followed by PE-conjugated anti-human Fcγ for the Fc-tagged proteins or anti-FLAG biotin followed by streptavidin APC for proteins with the FLAG tag. (C) L929 cells were incubated with biotin-labeled anti-CD9 mAb or biotin-labeled isotype control followed by streptavidin-APC. The median fluorescence intensity (MFI) of each treatment is shown.

Figure 2

Expression of heparan sulfate in bone marrow derived macrophages (BMDM) and binding of PSG23N-Fc to these cells. (A) BMDM derived from wild type or CD9-null mice and L929 cells were incubated with 1 μg of FITC-labeled anti-heparan sulfate 10E4 (anti-HS Ab) or FITC-labeled isotype control. (B) BMDM cells were incubated with 30 μg/ml of PSG23N-Fc or FLAG-Fc followed by anti-FLAG biotin and streptavidin APC.

Figure 3

PSG17 and PSG23 bind to Syndecans 1–4, Glypican-1 and to immobilized glycosaminoglycans. (A) Namalwa and Namalwa cells stably transfected with syndecans 1–4 or glypican-1 were incubated with 30 μg/ml of PSG17N-Fc, PSG23N-Fc or FLAG-Fc followed by PE-conjugated anti-human Fcγ. The median fluorescence intensity (MFI) of each treatment is shown. (B) 96-well NUNC plates were coated overnight with 200 μg/well of heparin, heparan sulfate, or chondroitin sulfate. After blocking, proteins were added to the wells. Following extensive washing, bound proteins were detected with HRP-conjugated anti-Fcγ Ab. All treatments were performed in quadruplicate and data is representative of three independent experiments. Statistical significance between the protein control and the PSG17N and 23N-treated wells was determined by two-tailed Student t-test and error bars represent the S.E.M. (C) PSG17N-Fc or PSG23N1AHisFLAG were applied to a 1 ml heparin-Sepharose column, after washing, the proteins were eluted with a NaCl gradient. Aliquots of each eluted fraction as well as the starting material (SM) and flow through (FT) were assessed by immunoblot analysis with HRP-conjugated anti-Fcγ Ab for PSG17N-Fc and HRP-conjugated anti-FLAG M2 mAb for PSG23N1AHisFLAG.

Figure 4

PSG17N binds to Sog9 cells expressing CD9 (Sog9-CD9) while PSG23 does not. (A)Sog9 cells were stably transfected with a plasmid encoding murine CD9. Expression of CD9 in the sorted cells was verified with anti-CD9 mAb. Sog9-CD9 cells were incubated with PSG17N-Fc (B) or PSG23N-Fc (C). Protein binding was detected with PE-conjugated anti-human Fcγ. (D) BHK-21 cells transfected with mCD9 or empty plasmid were added to Petri dishes coated with PSG17N, PSG23N or FLAG-Fc. After several washes, the cells bound to the dish were counted in three separate fields.

Figure 5

PSG-mutants and mapping of the CD9-binding site in the N1-domain of PSG17. (A) Identity between amino acids in the N1 domains of PSG17 and PSG23 is shown with a dash. Potential glycosylation sites are boxed. Highlighted amino acids indicate correspondence to the PSG23N1 sequence. “*” represents the K₃₅ to R substitution in PSG17 required to introduce a restriction site and “▲” indicates the N₅₂ to A mutation in mutant 6, which deletes one of the potential N-linked glycosylation sites in PSG17. The β-strands (A to G) are indicated with lines on the upper part of the figure. Total substitution refers to the number of amino acids changed from the wild type sequence. (B) Sog9 expressing murine CD9 were treated with 30 μg/ml of the mutants shown in part A, PSG17N-Fc or PSG23N-Fc followed by PE-conjugated anti-human Fcγ.

Figure 6

Differential binding of recombinant PSGs produced in CHO-K1 or HEK 293T cells to L929 cells. L929 cells were incubated with 30 μg/ml PSG17N-Fc (A) or PSG23N-Fc (B), which were generated in transiently transfected CHO-K1 or HEK 293T cells. After washing, the cells were incubated with PE-conjugated anti-human Fcγ. The median florescence intensity (MFI) of each treatment is shown.