Identification of novel serum proteins in a Japanese viper: homologs of mammalian PSP94

Three small serum proteins (SSP-1, -2, and -3), with molecular masses of 6.5-10kDa, were isolated from Habu (Trimeresurus flavoviridis) serum, and the amino acid sequences were determined by protein and cDNA analysis. Despite only limited sequence identity to any mammalian prostatic secretory protein of 94 amino acids (PSP94), all of the Cys residues in these SSPs were well conserved. SSPs are the first PSP94 family proteins to be identified in reptiles. SSP-1 and -3 weakly inhibited the proteolytic activity of a snake venom metalloproteinase. On the other hand, SSP-2 formed a tight complex with triflin, a snake venom-derived Ca(2+) channel blocker that suppresses the smooth muscle contraction. This suggests a role for SSP-2 in the self defense system of venomous snakes.     

Crystal Structure of Prostate Secretory Protein PSP94 Shows an Edge-to-Edge Association of two Monomers to Form a Homodimer

Several recent genome-wide association studies have linked the human MSMB gene, encoding prostate secretory protein of 94 residues (PSP94), with prostate cancer susceptibility. PSP94 is one of the most abundant proteins from prostatic secretions and a primary constituent of human semen. PSP94 suppresses tumor growth and metastasis, and its expression gradually decreases during progression of the prostate cancer. It is a rapidly evolving protein with homologues present in several species with 10 conserved cysteine residues. PSP94 homologues show high-affinity binding with different proteins from the cysteine-rich secretory protein family, some of which have been shown to be ion channel blockers. Here, we report the crystal structure of human PSP94 at 2.3 Å resolution. The structure shows that the amino and the carboxyl ends of the polypeptide chain are held in close proximity facing each other. A strong hydrogen bond between these ends, which are located respectively on the first and the last β-strands, leads to formation of an almost straight edge in PSP94 structure. Crystal structure shows that these edges from two PSP94 monomers associate in antiparallel fashion, leading to formation of a dimer. Our studies further show that dimers dissociate into monomers at acidic pH, possibly through distortion of the straight edge. Further, based on several observations, we propose that PSP94 binds to cysteine-rich secretory proteins and immunoglobulin G through the same edge, which is involved in the formation of PSP94 dimeric interface.     

High-affinity binding of seminal plasma PSP94 to human immunoglobulin is through the Fab domain

Prostate secretory protein of 94 amino acids (PSP94) is one of the major proteins present in human seminal plasma. We had earlier reported that PSP94 has the ability to bind to human IgG. The aims of the present study were to further delineate the PSP94–IgG interaction and to understand whether this could have any significance in sperm function. Direct binding of IgG fragments to PSP94 showed maximal binding with F(ab′)₂ followed by Fab, while Fc displayed least binding in ELISA. Binding kinetics of PSP94–IgG interaction using surface plasmon resonance (SPR) revealed high-affinity binding of IgG to PSP94 with a dissociation constant (K_D) of 8.8 × 10⁻¹¹ M. PSP94–IgG interaction was found to be through the Fab domains of IgG. Real-time interaction kinetics revealed association constants for binding of IgG, Fab, and F(ab′)₂ towards PSP94 to be of the same order but with altered dissociation constants. IgG and its F(ab′)₂ fragment once complexed to PSP94 demonstrated negligible dissociation, while dissociation rate of Fab fragment was 6.6 × 10⁻⁴. In silico molecular modeling of PSP94–IgG complex identified N- and C-terminal β-strands of PSP94 to be the most plausible region involved in IgG interaction. Immunofluorescence studies revealed that IgG bound to human spermatozoa predominantly in the tail region, which could be prevented when IgG was preincubated with PSP94. This study reports for the first time that IgG forms a high-affinity complex with PSP94 through its F(ab′)₂ domain and reveals the ability of PSP94 to prevent binding of IgG to spermatozoa.     

人PSP94全长cDNA的获得及PSP94-TNF~Δ融合蛋白的构建

利用ＲＴ－ＰＣＲ从人肥大前列腺组织钓取９４个氨基酸的人前列腺分泌蛋白（ＰＳＰ９４）全长ｃＤＮＡ,序列分析结果与文献报道的完全一致．将ＰＳＰ９４成熟肽与人ＴＮＦα衍生物（ＴＮＦΔ）通过Ｌｉｎｋｅｒ－ＳＡＰＧＴＰ在基因水平上融合成５′ＰＳＰ９４－ＴＮＦΔ,融合基因ＤＮＡ序列分析结果与设计的相符合．５′ＰＳＰ９４－ＴＮＦΔ在大肠杆菌中表达产物分子量约为３１ｋＤ,表达量约占菌体总蛋白量的３５％．以Ｌ９２９细胞和人前列腺癌细胞株ＰＣ－３为靶细胞进行细胞毒分析结果表明,５′ＰＳＰ９４－ＴＮＦΔ融合蛋白既具有ＴＮＦ的细胞毒活性,又具有对前列腺癌细胞ＰＣ－３的杀伤作用     

A model of the complex between human β-microseminoprotein and CRISP-3 based on NMR data

β-Microseminoprotein (MSP), a 10 kDa seminal plasma protein, forms a tight complex with cysteine-rich secretory protein 3 (CRISP-3) from granulocytes. The 3D structure of human MSP has been determined but there is as yet no 3D structure for CRISP-3. We have now studied the complex between human MSP and CRISP-3 with multidimensional NMR. ¹⁵N-HSQC spectra show substantial differences between free and complexed hMSP. Using several 3D-NMR spectra of triply labeled hMSP in complex with a recombinant N-terminal domain of CRISP-3, most of the backbone of hMSP could be assigned. The data show that only one side of hMSP, comprising β-strands 1, 4, 5, and 8 are affected by the complex formation, indicating that β-strands 1 and 8 form the main binding surface. Based on this we present a tentative structure for the hMSP-CRISP-3 complex using the known crystal structure of triflin as a model of CRISP-3.     

PSP94融合蛋白在大肠杆菌中的表达及其抗前列腺癌活性分析

在从成年人正常前列腺组织中获得人９４个氨基酸的前列腺分泌蛋白（ＰＳＰ９４）ｃＤＮＡ基础上,利用ＰＬ表达系统,实现了人ＰＳＰ９４成熟肽Ｎ 末端带有１９个外源氨基酸的融合蛋白在大肠杆菌中的表达。目的蛋白在细胞中主要以包涵体形式存在,表达量约占菌体总蛋白的３０％,分子量约为１６－５ｋＤ。表达产物在人前列腺癌细胞ＰＣ ３上活性分析表明,该融合蛋白能明显抑制前列腺癌细胞的生长。     

Human CRISP-3 binds serum α1B-glycoprotein across species

Background

CRISP-3 was previously shown to be bound to α₁B-glycoprotein (A1BG) in human serum/plasma. All mammalian sera are supposed to contain A1BG, although its presence in rodent sera is not well-documented. Since animal sera are often used to supplement buffers in experiments, in particular such that involve cell cultures, binding proteins present in sera might interfere in the experiments.

Methods

We examined sera from five different animal species for CRISP-3 binding proteins using gel filtration and ligand blotting. We developed a rapid method for isolation of proteins that bind to human CRISP-3 and identified the isolated proteins by mass spectrometry and N-terminal sequencing.

Results

We identified A1BG as a CRISP-3 binding protein in sera from cow, horse and rabbit. CRISP-3 bound kininogen 1 in mouse serum, whereas rat serum showed no CRISP-3 binding activity. In equine serum, we furthermore detected a possible CRISP, already bound to A1BG.

General significance

It seems to be a common mechanism that A1BGs bind CRISPs, also across species. Apart from the possible physiological implications hereof, complex binding of CRISPs by A1BG (and other proteins) may interfere with the detection and function of CRISPs, when these are studied in the presence of animal sera.     

Crystal structure of Brugia malayi venom allergen-like protein-1 (BmVAL-1), a vaccine candidate for lymphatic filariasis

Brugia malayi is a causative agent of lymphatic filariasis, a major tropical disease. The infective L3 parasite stage releases immunomodulatory proteins including the venom allergen-like proteins (VALs), which are members of the SCP/TAPS (Sperm-coating protein/Tpx/antigen 5/pathogenesis related-1/Sc7) superfamily. BmVAL-1 is a major target of host immunity with >90% of infected B. malayi microfilaraemic cases being seropositive for antibodies to BmVAL-1. This study is part of ongoing efforts to characterize the structures and functions of important B. malayi proteins. Recombinant BmVAL-1 was produced using a plant expression system, crystallized and the structure was solved by molecular replacement and refined to 2.1?Å, revealing the characteristic alpha/beta/alpha sandwich topology of eukaryotic SCP/TAPS proteins. The protein has more than 45% loop regions and these flexible loops connect the helices and strands, which are longer than predicted based on other parasite SCP/TAPS protein structures. The large central cavity of BmVAL-1 is a prototypical CRISP cavity with two histidines required to bind divalent cations. The caveolin-binding motif (CBM) that mediates sterol binding in SCP/TAPS proteins is large and open in BmVAL-1 and is N-glycosylated. N-glycosylation of the CBM does not affect the ability of BmVAL-1 to bind sterol in vitro. BmVAL-1 complements the in vivo sterol export phenotype of yeast mutants lacking their endogenous SCP/TAPS proteins. The in vitro sterol-binding affinity of BmVAL-1 is comparable with Pry1, a yeast sterol transporting SCP/TAPS protein. Sterol binding of BmVAL-1 is dependent on divalent cations. BmVAL-1 also has a large open palmitate-binding cavity, which binds palmitate comparably to tablysin-15, a lipid-binding SCP/TAPS protein. The central cavity, CBM and palmitate-binding cavity of BmVAL-1 are interconnected within the monomer with channels that can serve as pathways for water molecules, cations and small molecules.     

Sterol carrier protein‐2:Not just for cholesterol any more

Although sterol carrier protein-2 (SCP-2) mediates cholesterol esterification in L-cell fibroblasts and stimulates an accumulation of cholesterol in these cells, a potential role for SCP-2 in fatty acid uptake and trafficking has not been appreciated. Certainly, recent experiments have shown that SCP-2 binds fatty acids in vitro with an affinity similar to that observed for fatty acid binding proteins. Because of the ubiquitous tissue distribution of SCP-2, as opposed to the specific distribution of fatty acid binding proteins, as well as the need for fatty acid trafficking in all cells, I have recently proposed that SCP-2 is the universal fatty acid trafficking protein. This supposition is based on a number of observations made with L-cell fibroblasts expressing either the 13.2 kDa SCP-2 or the 15 kDa proSCP-2. In L-cells expressing the 13.2 kDa SCP-2, fluorescent fatty acid uptake was increased by 10–30% depending upon the probe used. In 15 kDa proSCP-2 expressing cells, fluorescent fatty acid uptake was increased 20–40% depending upon the probe used. However, only expression of the 15 kDa pro-SCP-2 increased the cytoplasmic diffusion of the fluorescent fatty acid. Expression of either protein increased the uptake of [³H]-oleic acid 1.9-fold compared to control, with targeting of [³H]-oleic acid for esterification into cholesteryl esters. The 13.2 kDa SCP-2 did target a significant amount of [³H]-oleic acid for esterification into the triacylglycerol pool. Expression of either protein markedly reduced total cellular phospholipid levels, however both proteins increased cholesteryl ester levels. Interestingly, expression of the 15 kDa proSCP-2 decreased ethanolamine plasmalogen levels with a concomitant increase in choline plasmalogen. Expression of both proteins increased PUFA content of the phospholipids, although this effect was greater in 15 kDa proSCP-2 expressing cells. Hence, expression of SCP-2 increased fatty acid uptake and targeted fatty acid to unique lipid pools, suggesting that SCP-2 may effectively serve as universal fatty acid binding and trafficking protein.