Identification of additional protein sequences containing only ZP-N

To investigate whether other proteins exist that contain only the N-terminal half of the ZP domain, we generated a profile hidden Markov model (HMM) of ZP-N to scan genomic and non-redundant sequence databases. This analysis identified three additional putative ZP-N-containing proteins, whose genes appear to be expressed (Table 1 and Fig. 1, underlined sequences). On the other hand, no proteins containing only ZP-C were found in a parallel search with a corresponding HMM profile. These observations suggest that, unlike ZP-N, ZP-C can be found exclusively within the context of a complete ZP domain.

Species	Protein name (accession number)	Amino acid number	HMM search*	Signal peptide†	Expression evidence	Reference(s)	Putative homolog(s)
C. elegans	F55A4.10 (AAL06028.2)	633	3.7e-13; 62.3; 31–126 (4)	0.999 (1–18); 11.327 (1–18)	ESTs (AU201804, CB402430), microarray (WormBase WBGene00018861)	[56]	-
D. melanogaster	Papillote/CG2467 (NP_727583.1)	963	4e-15; 69.0; 80–167 (4)	0.999 (1–32); 11.890 (1–32)	mRNA (AY862156), immunohisto-chemistry and Western blot [16], in situ hybridization (BDGP CG2467; [17])	[16, 17]	EAL32136.1 (D. pseudoobscura)
D. melanogaster	CG16798 (NP_610030.1)	561	9.9e-12; 57.7; 255–343 (4)	1.000 (1–28); 13.662 (1–28)	mRNA (AY122225), in situ hybridization (BDGP CG16798; [17])	[17]	SNAP00000007590 (A. gambiae)
D. melanogaster	CG10005 (NP_650137.3)	231	4.3e-17; 75.5; 59–151 (4)	0.999 (1–24); 6.625 (1–19)	mRNA (AY113516), microarray (BDGP CG10005)	-	EAL28621.1 (D. pseudoobscura), SNAP00000007531 (A. gambiae)
C. carpio	ZP1 § (CAA96573.1)	555	8.4e-29; 114.4; 381–482 (4)	0.999 (1–19); 6.833 (1–19)	MRNA (Z72492), Northern blot, in situ hydridization, immuno-histochemistry, Western blot [41]	[41]	-
M. musculus	Oosp1 (NP_579931.1)	202	3.2e-07; 42.7; 30–119 (4)	1.000 (1–21); 8.733 (1–21)	mRNA (AF420487), Northern blot, in situ hydridization [18]	[18, 19]	ENSRNOP00000028498 (R. norvegicus)
M. musculus	LOC225923/Oosp3 (NP_001028455.1)	194	1.2; 19.9; 28–117 (4)	1.000 (1–21); 8.275 (1–21)	mRNA (NM_001033283), RT-PCR, in situ hybridization [19]	[19]	-
H. sapiens	PLAC1 (NP_068568.1)	212	8.7e-11; 54.6; 29–119 (4)	0.999 (1–22); 7.539 (1–23)	MRNA (BC022335), Northern blot, in situ hydridizationm [11, 12, 15]	[11, 12, 15]	ENSPTRP00000038397 (P. troglodytes), ENSBTAP00000008260 (B. taurus), ENSCAFP00000027834 (C. familiaris), NP_001020065 (R. norvegicus), NP_062411.1 (M. musculus)
H. sapiens	LOC219990/Oosp2 (NP_776162.2)	158	0.0067; 28.4; 25–116 (4)	0.997 (1–17); 10.702 (1–17)	MRNA (NM_173801)	[19, 57]	ENSPTRP00000006381 (P. troglodytes) NP_001032723.1 (M. musculus)

* E-value; bit score; matched aa (number of Cys). Calibrated expectation values are relative to the NCBI non-redundant protein database (2308679 sequences at the time of the search).

^†SignalP probability (aa); SigCleave score (aa).

^§This protein is referred to as ZP2 in ref. 41. However, because it contains a trefoil domain immediately before the ZP domain, it should be regarded as a member of the ZP1 family.

Expression, purification and characterisation of recombinant ZP-N

To establish whether ZP-N is able to fold independently and investigate its biological role, we over-produced in recombinant form the PLAC1-homology region of the ZP domain of mouse ZP3. The 102-amino acid ZP-N fragment was expressed as an affinity sandwich [20], with E. coli maltose binding protein (MBP) fused to its N-terminus via a short linker and a polyhistidine tag (6his) fused to its C-terminus (Fig. 2A). MBP was chosen as a fusion partner since it is strictly monomeric in the presence of maltose [21, 22] and has either no or minimal interaction with the proteins to which it is fused, so that the stoichiometry of MBP fusion proteins is entirely determined by the properties of the non-MBP moieties [22, 23].

Using a bacterial strain that facilitates formation of disulfides by carrying trxB and gor mutations [24] and co-expressing modified versions of disulfide isomerase [24] and thioredoxin [25], significant amounts of MBP-ZP-N-6his were obtained that could be purified to homogeneity with a two-step affinity method (Fig. 2B, lane 2).

Although the fusion protein was soluble, as judged by ultracentrifugation at 100,000 g, it eluted in the void volume of 300 kDa molecular weight (M_r) cut-off size-exclusion columns, suggesting the presence of multimers. Analysis in the presence of ethylenedinitrilotetraacetic acid (EDTA) yielded identical elution profiles, excluding the possibility that trace amounts of Ni²⁺ ions could have leaked from the immobilised metal ion affinity chromatography (IMAC) column used during purification and caused non-specific protein aggregation by cross-linking multiple histidine tags.

Western blot analysis of purified MBP-ZP-N-6his revealed a band corresponding to monomeric protein and, in addition, a ladder of bands corresponding to dimers, tetramers etc. (i.e. 2n × M_r, with n = 1, 2, ...) (Fig. 2B). Although these multimers were much less abundant under reducing conditions, several lines of evidence suggest that this was due to more extensive denaturation of the ZP domain moiety of MBP-ZP-N-6his, rather than to the presence of spurious intermolecular disulfides. First, unlike the situation reported for other proteins [26], no bands were observed for trimeric, pentameric, etc. (i.e. (2n+1) × M_r) forms of MBP-ZP-N-6his (Fig. 2B). Second, as seen in the case of bands corresponding to the monomeric protein, dimeric and tetrameric MBP-ZP-N-6his also migrated differently under reducing and non-reducing conditions (Fig. 2B, compare lanes 2 and 3, and lanes 5 and 6, 7). Third, when samples were analysed by gel filtration under reducing conditions, most of the protein was still eluted in the void volume. Fourth, mass spectrometric analysis of proteolytic digests of dimeric MBP-ZP-N-6his did not reveal additional peaks compared to monomeric protein, whose spectra were consistent with native, intramolecular disulfides (ZP3 Cys 1 (aa 46)-Cys 4 (aa 139) and Cys 2 (aa 78)-Cys 3 (aa 98)) (Fig. 2C, D) [3, 6–10].

Structural analysis of recombinant ZP-N

Electron microscopy (EM) of negatively stained MBP-ZP-N-6his revealed that the protein assembles into long filaments (Fig. 3A) whose features are reminiscent of the helical structure described for full-length ZP domain proteins (Fig. 3B, C) [2, 3]. Moreover, a pattern was observed in immunolocalisation studies which suggests that dimeric MBP-ZP-N-6his is present as repeating units within filaments (Fig. 3D, E).

Sequence analysis

Calibrated profile HMMs for ZP-N and ZP-C were generated with HMMER 2.3.2 [44], using sequence databases derived from the Pfam [45] ZP domain protein family (PF00100) alignment. Sequences that were not complete within the amino acid range of interest were removed prior to HMM building. In the case of ZP-N, sequences that did not contain all conserved Cys 1–4 were also excluded, whereas conservation of Cys 5–8 was not explicitly imposed for inclusion of the more divergent ZP-C sequences. Profile HMMs were used to scan Ensembl [46] genome databases and the NCBI Entrez non-redundant protein database (~3800000 total sequences), and matching sequences were automatically extracted and submitted to BLAST [47], CD-SEARCH [48] and SMART [49]. Entries that were either partial (based on the alignment and annotation of matching BLAST sequences) or contained a complete ZP domain (as indicated by CD-SEARCH and/or SMART, as well as by their presence within both ZP-N and ZP-C matches) were filtered out, and remaining entries (~800 sequences) were individually analysed. Final acceptance criteria were high significance and completeness of the matches, as indicated by HMM E-values < 0.1 and extent of the alignment to HMM profiles (together with presence of conserved Cys 1–4 (ZP-N) or Cys 5–8 (ZP-C)), respectively. In addition, since both proteins with a complete ZP domain and PLAC1-like proteins are secreted, matches were accepted only if they also included a putative signal peptide (as predicted by SignalP [50] and EMBOSS SigCleave [51, 52]) which did not overlap with ZP domain sequence (as identified by CD-SEARCH and/or SMART). This analysis yielded 8 unique sequences containing only ZP-N, and no sequences containing only ZP-C (Table 1). An additional mouse sequence with E-value = 1.2 (protein LOC225923; accession number NP_001028455.1) was added to the ZP-N protein list on the basis of its significant similarity to proteins Oosp1 and LOC219990. BLAST and BLAT [53] searches of the mouse genome indicated that the genes encoding proteins Oosp1 and LOC225923, as well as the gene for a third protein (LOC225922; accession number NP_001032723.1) homologous to human LOC219990, are closely located on chromosome 19. The same cluster was independently identified in a recent study, in which LOC225922 and LOC225923 were renamed Oosp2 and Oosp3, respectively [19].

DNA constructs

A PCR fragment encoding aa 42–143 of mouse ZP3 protein was cloned between the Eco R1 and Xho 1 sites of vector pMBP4c, a derivative of plasmid pMBPL-/gp21(338–425) [54] that expresses a C-terminally histidine-tagged modified version of MBP under the control of T7 promoter/lac operator. A second vector, pLJDIS1, was generated from plasmids pBADΔSSdsbC [24] and pFÅ5 [25] to allow co-expression of a version of disulfide isomerase lacking a signal sequence (ΔSSdsbC) and a glutaredoxin-like thioredoxin variant with higher redox potential (TrxA(G33P, P34Y)), under the control of the arabinose promoter. All constructs were verified by DNA sequencing.

Protein expression and purification

For over-expression of MBP-ZP-N-6his, pMBP4c-mZP3(42–143) and pLJDIS1 were co-transformed into E. coli Origami B (DE3) (Novagen), carrying trxB and gor mutations. Although the trxB gor background was crucial to get partially soluble MBP-ZP-N-6his (the protein was completely insoluble in BL21 (DE3)), no significant improvement in solubility was observed upon co-expression of ΔSSdsbC or TrxA(G33P, P34Y). Nevertheless, we decided to still co-express both proteins, because they could be qualitatively important, as they were shown to significantly increase the activity of recombinant disulfide-rich proteins expressed in the cytoplasm of E. coli trxB gor strains [24]. Transformed cells were grown at 37°C in M9 medium containing 0.4% glucose, 15 μg/ml kanamycin, 12.5 μg/ml tetracyclin, 25 μg/ml chloramphenicol and 100 μg/ml carbenicillin. After reaching an optical density (OD_{595 nm}) of 0.5, they were shifted to 24°C for 30 min and pre-induced with 0.2% arabinose. 1 hr 30 min later, cells were induced with 0.1 mM isopropyl-β-D-thiogalactopyranoside and grown for an additional 25 hr at 24°C (final OD_{595 nm}~0.75). Bacteria were harvested by centrifugation and lysed with CelLytic B (Sigma). Soluble MBP-ZP-N-6his was purified by affinity chromatography, using Ni²⁺-charged HiTrap Chelating HP (Amersham Biosciences) and amylose resin (New England Biolabs) columns, followed by step-gradient ion exchange chromatography, using a Mono Q column (Amersham Biosciences). After dialysis against buffer F (10 mM Na-HEPES pH 8.0, 100 mM NaCl, 1 mM maltose, 1 mM NaN₃), the purified protein was concentrated to 16 mg/ml.

Western blotting

Immunoblot experiments were carried out by using BSA-free Penta•His monoclonal primary antibody (1:1000; QIAGEN) and goat anti-mouse horseradish peroxidase (HRP)-conjugated IgG (1:3000; ICN/Cappel), according to the manufacturers protocol. Chemiluminescent detection reactions were performed with Western Lightning Chemiluminescence Reagent Plus (Perkin Elmer).

Mass spectrometry

After SDS-PAGE under non-reducing conditions (with ~20 μg MBP-ZP-N-6his/lane), gel spots were excised and alkylated with 30 mM iodoacetamide in 100 mM Tris-HCl pH 6.8 for 30 min at room temperature. The liquid was removed and samples were prepared for digestion by washing twice with 100 ml 50 mM Tris-HCl pH 6.8/30% acetonitrile (ACN) for 20 min with shaking, then with 100% ACN for 1–2 min. After removing the washes, gel pieces were dried for 30 min in a Speed-Vac concentrator. Individual gel pieces were digested by adding 80 μg modified trypsin or chymotrypsin (sequencing grade, Roche Molecular Biochemicals) in 13–15 ml 25 mM Tris-HCl pH 6.8 and leaving overnight at room temperature. Peptides were extracted with 2 × 50 ml 50% ACN/2% trifluoroacetic acid (TFA) and the combined extracts were divided in half, then dried. One half of the digest was dissolved in matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) matrix for immediate mass spectrometric analysis, and the other half was reduced by adding 20 mM dithiothreitol (DTT) in 100 mM Tris-HCl pH 8.5. After 30 min at 50°C, the reduced digest was cooled to room temperature and desalted with a C18 ZipTip (Millipore), using 50% ACN to elute the peptides. The eluate was dried and dissolved in MALDI-TOF-MS matrix for analysis. Matrix solution was prepared by making a 10 mg/ml solution of 4-hydroxy-α-cyanocinnamic acid in 50% ACN/0.1% TFA. The dried digest was dissolved in 3 ml matrix solution and 0.7 ml was spotted onto the sample plate. If the sample was not previously desalted, the dried spot was washed twice with water. MALDI mass spectrometric analysis was performed on the digest using a Voyager DE-Pro mass spectrometer (Applied Biosystems) in the linear mode. Spectra were analysed both manually and with MS-Screener [55] and MS-Compare (LJ, unpublished). Since all samples were alkylated prior to digestion, unmodified free Cys-containing peptides identified under non-reducing conditions (Fig. 2C) resulted from laser-induced breakage of disulfides. Furthermore, it appeared that essentially all Cys residues of purified MBP-ZP-N-6his were involved in disulfides. Unlike the case of the Cys 2-Cys 3 disulfide bridge (Fig. 2C), a peak corresponding to a linkage between peptides containing Cys 1 and Cys 4 could not be identified under non-reducing conditions; however, existence of the latter bridge could be clearly inferred by appearance (or marked increase in the intensity) of peaks corresponding to peptides containing unmodified free Cys 1 and Cys 4 upon reduction of the sample (compare Fig. 2C and 2D). This was further supported by a corresponding increase in the intensity of a peak corresponding to the C-terminal tag, which closely follows Cys 4 in the sequence of MBP-ZP-N-6his (Fig. 2C, D). MALDI-TOF-MS analyses of chymotrypsin-digested monomeric protein as well as trypsin-digested dimeric MBP-ZP-N-6his were also consistent with intramolecular 1–4, 2–3 disulfides.

Size-exclusion chromatography

Gel filtration experiments were performed on both FPLC and HPLC systems, using a HiPrep 16/60 Sephacryl S-300 HR column (~300 kDa M_r cut-off; Amersham Biosciences) and a Bio-Sil SEC-250-5 column (~300 kDa M_r cut-off; Bio-Rad), respectively. Running solutions were buffer F (non-reducing conditions) or buffer F + 10 mM DTT (reducing conditions). Additional runs were performed by pre-incubating purified MBP-ZP-N-6his with 10 mM EDTA pH 8.0 for 1 hr at 4°C, before analysis using 10 mM Na-HEPES pH 8.0, 1 mM EDTA as running buffer.

Electron microscopy

For morphological observation, material was negatively stained by applying a drop of solution (final concentration 1 mg/ml) directly onto a 300-mesh formvar-carbon coated nickel grid (Electron Microscopy Sciences), which was allowed to remain for approximately 30 seconds, after which excess solution was removed. A drop of 1% aqueous uranyl acetate was then added onto the grid and allowed to remain for an additional 30 seconds, after which excess solution was removed and the grids allowed to dry. For immunogold localisation, equal volumes of protein (1 mg/ml) and anti-MBP monoclonal primary antibody (1:300; New England Biolabs) diluted in Tris-buffered saline-Tween-20 solution (TBS-T) were allowed to incubate for two hours at room temperature. Goat anti-mouse H&L(Fab2') 10 nm gold-conjugated secondary antibody (1:30/TBS-T, EMS) was added directly to the solution and allowed to incubate for two hours at room temperature. A 300-mesh formvar-carbon coated nickel grid was then immersed and allowed to remain for approximately 30 seconds, after which it was removed and excess solution was removed. Negative contrast staining followed the above-described method. Material was imaged on a Jeol 1200EX electron microscope equipped with an Advanced Imaging Technologies digital camera. Images were imported into Photoshop CS2 (Adobe Systems Inc.) where they were sized and optimised for contrast and brightness.