Cysteine-rich secretory proteins (CRISP) and their role in mammalian fertilization

Epididymal protein CRISPI is a member of the CRISP (Cysteine-RIch Secretory proteins) family and is involved in sperm-egg fusion through its interaction with complementary sites on the egg surface. Results from our laboratory have shown that this binding ability resides in a 12-amino-acid region corresponding to a highly conserved motif of the CRISP family, named Signature 2 (S2). In addition to this, our results revealed that CRISP1 could also be involved in the previous step of sperm binding to the zona pellucida, identifying a novel role for this protein in fertilization. As another approach to elucidate the participation of CRISP1 in fertilization, a mouse line containing a targeted disruption of CRISP1 was generated. Although CRISP1-deficient mice exhibited normal fertility, CRISP1-defficient sperm presented a decreased level of protein tyrosine phosphorylation during capacitation, and an impaired ability to fertilize both zona-intact and zona-free eggs in vitro, confirming the proposed roles for the protein in fertilization. Evidence obtained in our laboratory indicated that testicular CRISP2 would also be involved in sperm-egg fusion. Competition assays between CRISP1 and CRISP2, as well as the comparison of their corresponding S2 regions, suggest that both proteins bind to common complementary sites in the egg. Together, these results suggest a functional cooperation between CRISP1 and CRISP2 to ensure the success of fertilization.     

Matriptase-2 (TMPRSS6) is directly up-regulated by hypoxia inducible factor-1: identification of a hypoxia-responsive element in the TMPRSS6 promoter region

The type II transmembrane serine protease matriptase-2 (TMPRSS6) down-regulates the expression of hepcidin, the main regulator of systemic iron homeostasis, and increases in this way iron plasma levels. Matriptase-2 is up-regulated under hypoxic conditions, providing a new link between hypoxia signaling and iron homeostasis. In this study, we have characterized the TMPRSS6 promoter region and identified a functional hypoxia-responsive element (HRE). Mutations of the hypoxia inducible factor (HIF)-binding site located within the HRE abrogate HIF-1α-dependent induction of TMPRSS6 expression. The action of HIF-1α on TMPRSS6 promoter activity reveals a new regulative element for the suppression of hepcidin synthesis.     

Preferential expression of cystein-rich secretory protein-3 (CRISP-3) in chronic pancreatitis

BACKGROUND: Chronic pancreatitis (CP) is a progressive inflammatory process resulting in exocrine and endocrine pancreatic insufficiency in advanced stages. Cysteine-rich secretory protein (CRISP-3) has been identified as a defense-associated molecule with predominant expression in the salivary gland, pancreas and prostate. AIMS: In this study, we investigated CRISP-3 expression in normal pancreatic tissues, chronic pancreatitis tissues, pancreatic cancer tissues and pancreatic cancer cell lines, as well as in other gastrointestinal organs. MATERIALS AND METHODS: 15 normal pancreatic tissues, 14 chronic pancreatitis tissues and 14 pancreatic cancer tissues as well as three pancreatic cancer cell lines were analyzed. Moreover, hepatocellular carcinoma and esophageal, stomach and colon cancers were also analyzed together with the corresponding normal controls. RESULTS: CRISP-3 was expressed at moderate to high levels in chronic pancreatitis tissues and at moderate levels in pancreatic cancer tissues but at low levels in normal pancreatic tissues, and was absent in three pancreatic cancer cell lines. CRISP-3 expression was below the level of detection in all cancerous gastrointestinal tissues and in all normal tissues except 2 of 16 colon tissue samples. CRISP-3 mRNA signals and immunoreactivity were strongly present in the cytoplasm of degenerating acinar cells and in small proliferating ductal cells in CP tissues and CP-like lesions in pancreatic cancer tissues. In contrast, CRISP-3 expression was weak to absent in the cytoplasm of cancer cells as well as in acinar cells and ductal cells in pancreatic cancer tissues and normal pancreatic tissues. CONCLUSION: These results reveal that the distribution of CRISP-3 in gastrointestinal tissues is predominantly in the pancreas. High levels of CRISP-3 in acinar cells dedifferentiating into small proliferating ductal cells in CP and CP-like lesions in pancreatic cancer suggests a role of this molecule in the pathophysiology of CP.     

Secretor genotype (FUT2 gene) is strongly associated with the composition of Bifidobacteria in the human intestine

Intestinal microbiota plays an important role in human health, and its composition is determined by several factors, such as diet and host genotype. However, thus far it has remained unknown which host genes are determinants for the microbiota composition. We studied the diversity and abundance of dominant bacteria and bifidobacteria from the faecal samples of 71 healthy individuals. In this cohort, 14 were non-secretor individuals and the remainders were secretors. The secretor status is defined by the expression of the ABH and Lewis histo-blood group antigens in the intestinal mucus and other secretions. It is determined by fucosyltransferase 2 enzyme, encoded by the FUT2 gene. Non-functional enzyme resulting from a nonsense mutation in the FUT2 gene leads to the non-secretor phenotype. PCR-DGGE and qPCR methods were applied for the intestinal microbiota analysis. Principal component analysis of bifidobacterial DGGE profiles showed that the samples of non-secretor individuals formed a separate cluster within the secretor samples. Moreover, bifidobacterial diversity (p<0.0001), richness (p<0.0003), and abundance (p<0.05) were significantly reduced in the samples from the non-secretor individuals as compared with those from the secretor individuals. The non-secretor individuals lacked, or were rarely colonized by, several genotypes related to B. bifidum, B. adolescentis and B. catenulatum/pseudocatenulatum. In contrast to bifidobacteria, several bacterial genotypes were more common and the richness (p<0.04) of dominant bacteria as detected by PCR-DGGE was higher in the non-secretor individuals than in the secretor individuals. We showed that the diversity and composition of the human bifidobacterial population is strongly associated with the histo-blood group ABH secretor/non-secretor status, which consequently appears to be one of the host genetic determinants for the composition of the intestinal microbiota. This association can be explained by the difference between the secretor and non-secretor individuals in their expression of ABH and Lewis glycan epitopes in the mucosa.     

A polymorphism within the equine CRISP3 gene is associated with stallion fertility in Hanoverian warmblood horses

Fertility of stallions is of high economic importance, especially for large breeding organisations and studs. Breeding schemes with respect to fertility traits and selection of stallions at an early stage may be improved by including molecular genetic markers associated with traits. The genes coding for equine cysteine-rich secretory proteins (CRISPs) are promising candidate genes because previous studies have shown that CRISPs play a role in the fertilising ability of male animals. We have previously characterised the three equine CRISP genes and identified a non-synonymous polymorphism in the CRISP1 gene. In this study, we report one non-synonymous polymorphism in the CRISP2 gene and four non-synonymous polymorphisms in the CRISP3 gene. All six CRISP polymorphisms were genotyped in 107 Hanoverian breeding stallions. Insemination records of stallions were used to analyse the association between CRISP polymorphisms and fertility traits. Three statistical models were used to evaluate the influence of single mutations, genotypes and haplotypes of the polymorphisms. The CRISP3 AJ459965:c.+622G>A SNP leading to the amino acid substitution E208K was significantly associated with the fertility of stallions. Stallions heterozygous for the CRISP3 c.+622G>A SNP had lower fertility than homozygous stallions (P = 0.0234). The pregnancy rate per cycle in these stallions was estimated to be approximately 7% lower than in stallions homozygous at this position.     

Bone morphogenetic protein-3b (BMP-3b) gene expression is correlated with differentiation in rat calvarial osteoblasts

BMP-3b (also called GDF-10) is a novel BMP-3-related protein recently discovered in rat femur tissue. Gene expression of BMP-3b in osteoblastic cells and its regulation by prolonged culture, BMP-2 and transforming growth factor beta1 (TGF-beta1) were examined. The BMP-3b gene was highly expressed in rat osteoblasts obtained from calvarial bones but not in the osteoblastic cell lines (MC3T3-E1 and U2-OS). BMP-3b mRNA increased during osteoblastic differentiation in prolonged culture and was associated with increased alkaline phosphatase (ALPase) activity. When BMP-2, an enhancer of ALPase activity, was added to the primary osteoblast culture, BMP-3b mRNA increased 6.9-fold after 24 h. In contrast, TGF-beta1 treatment, which suppresses ALPase activity, rapidly and completely inhibited gene expression of BMP-3b. The regulation of BMP-3 mRNA differed from that of BMP-3b, even though both proteins share 81% identity. These findings indicate that BMP-3b gene expression is regulated by osteoblastic differentiation and BMP-3b functions in highly differentiated osteoblasts.     

Biochemical and conformational characterisation of HSP-3, a stallion seminal plasma protein of the cysteine-rich secretory protein (CRISP) family

HSP-3 is a member of the cysteine-rich secretory protein (CRISP) family from stallion seminal plasma. We report a large-scale purification protocol for native HSP-3. This protein is a non-glycosylated polypeptide chain with a pI of 8–9 and an isotope-averaged molecular mass of 24 987±3 Da. The molecular mass of HSP-3, determined by equilibrium sedimentation, is 26 kDa, showing that the protein exists in solution as a monomer. The concentration of HSP-3 in the seminal plasma of different stallions ranged from 0.3 to 1.3 mg/ml. On average, 0.9–9 million HSP-3 molecules/cell coat the postacrosomal and mid-piece regions of an ejaculated, washed stallion spermatozoon, suggesting a role in sperm physiology. Conformational characterisation of purified HSP-3 was assessed by combination of circular dichroism and Fourier-transform infrared spectroscopies and differential scanning microcalorimetry. Based on secondary structure assignment, HSP-3 may belong to the α+β class of proteins. Thermal denaturation of HSP-3 is irreversible and follows a non-two state transition characterised by a T_m of 64°C, an enthalpy change of 75 kcal/mol, and a van 't Hoff enthalpy of 184 kcal/mol. Analysis of the spectroscopic and calorimetric data indicates the occurrence of aggregation of denatured HSP-3 molecules and suggests the monomer as the cooperative unfolding unit.     

A novel protein,sperm head and tail associated protein (SHTAP), interacts with cysteine‐rich secretory protein 2 (CRISP2) during spermatogenesis in the mouse

Background information. CRISP2 (cysteine‐rich secretory protein 2) is a sperm acrosome and tail protein with the ability to regulate Ca²⁺ flow through ryanodine receptors. Based on these properties, CRISP2 has a potential role in fertilization through the regulation of ion signalling in the acrosome reaction and sperm motility. The purpose of the present study was to determine the expression, subcellular localization and the role in spermatogenesis of a novel CRISP2‐binding partner, which we have designated SHTAP (sperm head and tail associated protein). Results. Using yeast two‐hybrid screens of an adult testis expression library, we identified SHTAP as a novel mouse CRISP2‐binding partner. Sequence analysis of all Shtap cDNA clones revealed that the mouse Shtap gene is embedded within a gene encoding the unrelated protein NSUN4 (NOL1/NOP2/Sun domain family member 4). Five orthologues of the Shtap gene have been annotated in public databases. SHTAP and its orthologues showed no significant sequence similarity to any known protein or functional motifs, including NSUN4. Using an SHTAP antiserum, multiple SHTAP isoforms (～20–87 kDa) were detected in the testis, sperm, and various somatic tissues. Interestingly, only the ～26 kDa isoform of SHTAP was able to interact with CRISP2. Furthermore, yeast two‐hybrid assays showed that both the CAP (CRISP/antigen 5/pathogenesis related‐1) and CRISP domains of CRISP2 were required for maximal binding to SHTAP. SHTAP protein was localized to the peri‐acrosomal region of round spermatids, and the head and tail of the elongated spermatids and sperm tail where it co‐localized with CRISP2. During sperm capacitation, SHTAP and the SHTAP—CRISP2 complex appeared to be redistributed within the head. Conclusions. The present study is the first report of the identification, annotation and expression analysis of the mouse Shtap gene. The redistribution observed during sperm capacitation raises the possibility that SHTAP and the SHTAP—CRISP2 complex play a role in the attainment of sperm functional competence.     

First evidence of uncoupling protein-2 (UCP-2) and -3 (UCP-3) gene expression in piglet skeletal muscle and adipose tissue

Uncoupling proteins (UCPs) facilitate proton transport inside the mitochondria and decrease the proton gradient, leading to heat production. Until now, the presence of UCP1 or other UCP homologs had not been detected in tissues of pig, a species where evidence for the presence of brown adipose tissue has only been provided in 2-3 month old animals. In the light of the improving knowledge on the UCPs family, we decided to examine both UCP2 and UCP3 mRNA expression in piglet skeletal muscle and adipose tissue. Using RT-PCR we have successfully cloned a partial UCP2 sequence and a complete UCP3 cDNA. UCP3's open reading frame (936bp) shares 90, 89 and 85% similarity with bovine, human and rat UCP3 nucleotide sequences, respectively. In 3-5 day old piglets, these genes are expressed in adipose tissue and in both longissimus thoracis (LT) and rhombo?deus (RH) muscles, without any effect of muscle metabolic type. This is in good agreement with the measurement of the same membrane potential in mitochondria isolated from both types of muscles. In triiodothyronine-treated piglets, UCP3 mRNA is more expressed in LT than in RH muscle. These genes may be involved in the control of the energy metabolism of the piglet.     

Molecular analysis of the TMPRSS3 gene in Moroccan families with non-syndromic hearing loss

Autosomal recessive non-syndromic hearing impairment (ARNSHI) is the most common type of inherited hearing impairment, accounting for approximately 80% of inherited prelingual hearing impairment. Hearing loss is noted to be both phenotypically and genetically heterogeneous. Mutations in the TMPRSS3 gene, which encodes a transmembrane serine protease, are known to cause autosomal recessive non-syndromic hearing impairment DFNB8/10. In order to elucidate if the TMPRSS3 gene is responsible for ARNSHI in 80 Moroccan families with non-syndromic hearing impairment, the gene was sequenced using DNA samples from these families. Nineteen TMPRSS3 variants were found, nine are located in the exons among which six are missense and three are synonymous. The 10 remaining variations are located in non-coding regions. Missense variants analysis show that they do not have a significant pathogenic effect on protein while pathogenicity of some variant remains under discussion. Thus we show that the TMPRSS3 gene is not a major contributor to non-syndromic deafness in the Moroccan population.     

Group IID heparin-binding secretory phospholipase A(2) is expressed in human colon carcinoma cells and human mast cells and up-regulated in mouse inflammatory tissues.

Group IID secretory phospholipase A(2) (sPLA(2)-IID), a heparin-binding sPLA(2) that is closely related to sPLA(2)-IIA, augments stimulus-induced cellular arachidonate release in a manner similar to sPLA(2)-IIA. Here we identified the residues of sPLA(2)-IID that are responsible for heparanoid binding, are and therefore essential for cellular function. Mutating four cationic residues in the C-terminal portion of sPLA(2)-IID resulted in abolition of its ability to associate with cell surface heparan sulfate and to enhance stimulus-induced delayed arachidonate release, cyclooxygenase-2 induction, and prostaglandin generation in 293 cell transfectants. As compared with several other group II subfamily sPLA(2)s, which were equally active on A23187- and IL-1-primed cellular membranes, sPLA(2)-IID showed apparent preference for A23187-primed membranes. Several human colon carcinoma cell lines expressed sPLA(2)-IID and sPLA(2)-X constitutively, the former of which was negatively regulated by IL-1. sPLA(2)-IID, but not other sPLA(2) isozymes, was expressed in human cord blood-derived mast cells. The expression of sPLA(2)-IID was significantly altered in several tissues of mice with experimental inflammation. These results indicate that sPLA(2)-IID may be involved in inflammation in cell- and tissue-specific manners under particular conditions.     

Identification of thioredoxin-binding protein-2/vitamin D(3) up-regulated protein 1 as a negative regulator of thioredoxin function and expression.

Recent works have shown the importance of reduction/oxidation (redox) regulation in various biological phenomena. Thioredoxin (TRX) is one of the major components of the thiol reducing system and plays multiple roles in cellular processes such as proliferation, apoptosis, and gene expression. To investigate the molecular mechanism of TRX action, we used a yeast two-hybrid system to identify TRX-binding proteins. One of the candidates, designated as thioredoxin-binding protein-2 (TBP-2), was identical to vitamin D(3) up-regulated protein 1 (VDUP1). The association of TRX with TBP-2/VDUP1 was observed in vitro and in vivo. TBP-2/VDUP1 bound to reduced TRX but not to oxidized TRX nor to mutant TRX, in which two redox active cysteine residues are substituted by serine. Thus, the catalytic center of TRX seems to be important for the interaction. Insulin reducing activity of TRX was inhibited by the addition of recombinant TBP-2/VDUP1 protein in vitro. In COS-7 and HEK293 cells transiently transfected with TBP-2/VDUP1 expression vector, decrease of insulin reducing activity of TRX and diminishment of TRX expression was observed. These results suggested that TBP-2/VDUP1 serves as a negative regulator of the biological function and expression of TRX. Treatment of HL-60 cells with 1alpha, 25-dihydroxyvitamin D(3) caused an increase of TBP-2/VDUP1 expression and down-regulation of the expression and the reducing activity of TRX. Therefore, the TRX-TBP-2/VDUP1 interaction may be an important redox regulatory mechanism in cellular processes, including differentiation of myeloid and macrophage lineages.     

Autoantigen 1 of the guinea pig sperm acrosome is the homologue of mouse Tpx-1 and human TPX1 and is a member of the cysteine-rich secretory protein (CRISP) family

We have cloned and sequenced cDNAs encoding autoantigen 1 (AA1), a testis-specific protein and the major autoantigen of the guinea pig sperm acrosome. The cDNA predicts a precursor protein of 244 amino acids including a 21 amino acid hydrophobic, secretory signal sequence. The mature polypeptide is predicted to have a molecular mass of 24,891 Daltons which agrees with the experimentally determined molecular weight of 25,000. Consistent with previous studies demonstrating that AA1 is not a glycoprotein, the predicted amino acid sequence contained no canonical sites for N-linked glycosylation. Comparison with other sequences showed that AA1 is the guinea pig homologue of the testis-specific protein Tpx-1 in mice and TPX1 in humans. AA1 also showed significant amino acid sequence homology with other cysteine-rich secretory proteins (CRISP's): rat and mouse acidic epididymal glycoproteins (AEG; also known as proteins D/E in rats) and helothermine, a toxin from the Mexican beaded lizard. In addition, AA1 had a lesser degree of homology with antigen 5 (vespid wasp venom), PR-1 (a plant pathogenesis related protein), and GliPR (a protein identified in human gliomas). Northern analysis of RNA from purified guinea pig spermatogenic cells showed that a 1.5 kb message was first detected in pachytene spermatocytes, was strongest in round spermatids, and was detected at a low level in condensing spermatids. Immunoblot analysis and metabolic labeling data of AA1 in spermatogenic cells showed that the protein was synthesized as early as the pachytene spermatocyte stage of spermatogenesis. Thus, the patterns of AA1 mRNA and protein expression during spermatogenesis are similar to the expression of other acrosomal mRNAs and proteins that are first detected meiotically. © 1996 Wiley-Liss, Inc.     

Structural organization and mutational analysis of the human uncoupling protein-2 (hUCP2) gene

Uncoupling proteins (UCPs) are mitochondrial membrane transporters which are involved in dissipating the proton electrochemical gradient thereby releasing stored energy as heat. This implies a major role of UCPs in energy metabolism and thermogenesis which when deregulated are key risk factors for the development of obesity and other eating disorders. From the three different human UCPs identified so far by gene cloning both UCP2 and UCP3 were mapped in close proximity (75-150 kb) to regions of human chromosome 11 (11q13) that have been linked to obesity and hyperinsulinaemia. At the amino acid level hUCP2 has about 55% identity to hUCP1 while hUCP3 is 71% identical to hUCP2. In this study we have deduced the genomic structure of the human UCP2 gene by PCR and direct sequence analysis. The hUCP2 gene spans over 8.7 kb distributed on 8 exons. The localization of the exon/intron boundaries within the coding region matches precisely that of the hUCP1 gene and is almost conserved in the recently discovered hUCP3 gene as well. The high degree of homology at the nucleotide level and the conservation of the exon /intron boundaries among the three UCP genes suggests that they may have evolved from a common ancestor or are the result from gene duplication events. Mutational analysis of the hUCP2 gene in a cohort of 172 children (aged 7 - 13) of Caucasian origin revealed a polymorphism in exon 4 (C to T transition at position 164 of the cDNA resulting in the substitution of an alanine by a valine at codon 55) and an insertion polymorphism in exon 8. The insertion polymorphism consists of a 45 bp repeat located 150 bp downstream of the stop codon in the 3'-UTR. The allele frequencies were 0.63 and 0.37 for the alanine and valine encoded alleles, respectively, and 0.71 versus 0.29 for the insertion polymorphism. The allele frequencies of both polymorphisms were not significantly elevated in a subgroup of 25 children characterized by low Resting Metabolic Rates (RMR). So far a direct correlation of the observed genotype with (RMR) and Body Mass Index (BMI) was not evident. Expression studies of the wild type and mutant forms of UCP2 should clarify the functional consequences these polymorphisms may have on energy metabolism and body weight regulation.