The epididymal protease inhibitor (Eppin) abounds in human semen and on the surface of human spermatozoa, specifically produced by the testis and epididymis. Recombinant Eppin has effected infertility in the immunized monkey and promises to be an effective vaccine for human immunocontraception. This article reviews the advances in the studies of Eppin gene and protein construction and its molecular mechanism of causing immunologic infertility and regulating PSA hydrolysis of Semenogelin.
Animals ; Humans ; Infertility, Male ; immunology ; Male ; Mice ; Primates ; Proteinase Inhibitory Proteins, Secretory ; chemistry ; genetics ; immunology ; physiology ; Seminal Vesicle Secretory Proteins ; physiology ; Vaccines, Contraceptive

The aim of this study is to develop monoclonal antibody against human hepatocyte growth factor activator inhibitor 1 (HAI-1) for future study of HAI-1. The cDNA fragments of human hepatocyte growth factor activator inhibitor 1 (HAI-1) were subcloned to construct GST-HAI-1 fusion protein expression vectors. The vectors were transformed into E. coli and fusion protein expression was induced by IPTG. The GST-HAI-1 fusion proteins were separated on preparative SDS-PAGE and recovered by electroelution, and used to immunize BALB/c mice. Hybridomas producing monoclonal antibodies against human HAI-1 were prepared by cell fusion technique and characterized by ELISA, Western Blot and immunohistochemical staining. One hybridoma cell line, ZMC6, was obtained, which produces specific antibody against the expressed GST-HAI-1 fusion protein. The monoclonal antibody recognizes both the membrane-type and secretory-type HAI-1 proteins of colorectal tissue. The successful development of anti-HAI-1 antibody provides a powerful tool for further investigation on HAI-1's function.
Animals ; Antibodies, Monoclonal ; immunology ; Blotting, Western ; Glutathione Transferase ; genetics ; Immunohistochemistry ; Mice ; Mice, Inbred BALB C ; Proteinase Inhibitory Proteins, Secretory ; analysis ; genetics ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; immunology

BACKGROUNDEppin (epididymis protease inhibitor) appears to play an important role in primate fertility. However, the function of Eppin and its antibody in men and its relationship with men's infertility are poorly studied. To reveal the significance and possibility of detection of anti-Eppin antibody in clinical infertilty cases, we developed an Escherichia coli expression system for the expression of biologically active human Eppin.

METHODSThe human Eppin gene was cloned into PET-28a( )+ vector after induction with 0.5 mmol/L isopropy-beta-D-thiogalactoside (IPTG) at 26 degrees C for 4 hours, and the expressed fusion protein His6-Eppin was purified by Ni2+ affinity chromatography. Afterwards, six female 8-week-old Balb/c mice were immunized with purified His6-Eppin for three weeks. Their sera were collected and polyclonal antibodies against His6-Eppin were purified, all of which were further verified by Western-blot and immunofluorescence analysis.

RESULTSAbout 18.33 mg His6-Eppin was obtained from 1-L flask culture. The produced polyclonal antibodies against His6-Eppin recognized the Eppin protein both in human epididymis and in HEK293T cells by over-expression of the recombinant human Eppin.

CONCLUSIONThe purified His6-Eppin protein has biological activity, which might be a candidate for clinical diagnosis of infertility and development of male immuno-contraceptive agents.

Animals ; Escherichia coli ; genetics ; Female ; Fluorescent Antibody Technique ; Humans ; Mice ; Mice, Inbred BALB C ; Proteinase Inhibitory Proteins, Secretory ; immunology ; isolation & purification ; Recombinant Fusion Proteins ; immunology ; isolation & purification

Hespintor is an unknown function protein that was got from hepatoblastoma cell lines HepG2 by suppression subtractive hybridization technique (SSH), sequence analysis showed that the protein is a new member of secretory type of Kazal type serine protease inhibitor (Serpin) family, and has high homology with esophageal cancer related gene 2 (ECRG2). The coding sequence of Hespintor's Kazal domain was subcloned into prokaryotic expression vector pET-40b(+), then transformed into Rosetta (DE3). A recombinant protein about 42 kDa in the form of inclusion body was optimization expressed by inducing with 0.25 mmol/L IPTG, 30 degrees C for 5 h. and its specificity was confirmed via Western blotting. The recombinant protein was purified by metal chelate affinity chromatography (MCAC) and anion-exchange chromatography. The preliminary experimental result showed that the recombinant protein can inhibit trysin hydrolysis activity specifically. The result clearly demonstrated that Hespintor, as a novel member of Serpin, would be valuable in developing anti-tumor agents.
Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Hep G2 Cells ; Humans ; Proteinase Inhibitory Proteins, Secretory ; Recombinant Proteins ; biosynthesis ; genetics ; Serine Peptidase Inhibitors, Kazal Type ; Serine Proteinase Inhibitors ; biosynthesis ; classification ; genetics

AIMTo study the molecular mechanism of epididymal protease inhibitor (Eppin) modulating the process of prostate specific antigen (PSA) digesting semenogelin (Sg).

METHODSHuman Sg cDNA (nucleotides 82-849) and Eppin cDNA (nucleotides 70-723) were generated by polymerase chain reaction (PCR) and cloned into pET-100D/TOPO. Recombinant Eppin and Sg (rEppin and rSg) were produced by BL21 (DE3). The association of Eppin with Sg was studied by far-western immunoblot and radioautography. In vitro the digestion of rSg by PSA in the presence or absence of rEppin was studied. The effect of anti-Q20E (N-terminal) and C-terminal of Eppin on Eppin-Sg binding was monitored.

RESULTSEppin binds Sg on the surface of human spermatozoa with the C-terminal of Eppin (amino acids 75-133). rSg was digested with PSA and many low molecular weight fragments were produced. When rEppin is bound to rSg, then digested by PSA, incomplete digestion and a 15-kDa fragment results. Antibody binding to the N-terminal of rEppin did not affect rSg digestion. Addition of antibodies to the C-terminal of rEppin inhibited the modulating effect of rEppin.

CONCLUSIONEppin protects a 15-kDa fragment of rSg from hydrolysis by PSA.

Animals ; Antibodies ; pharmacology ; Autoradiography ; Humans ; Hydrolysis ; Male ; Prostate-Specific Antigen ; metabolism ; Proteinase Inhibitory Proteins, Secretory ; genetics ; immunology ; metabolism ; Rabbits ; Recombinant Proteins ; genetics ; metabolism ; Semen ; cytology ; metabolism ; Seminal Vesicle Secretory Proteins ; metabolism ; Spermatozoa ; metabolism

X-linked ichthyosis (XLI) is a recessively inherited ichthyosis. Skin barrier function of XLI patients reported in Western countries presented minimally abnormal or normal. Here, we evaluated the skin barrier properties and a skin barrier-related gene mutation in 16 Korean XLI patients who were diagnosed by fluorescence in situ hybridization and array comparative genomic hybridization analysis. Skin barrier properties were measured, cytokine expression levels in the stratum corneum (SC) were evaluated with the tape stripped specimen from skin surface, and a genetic test was done on blood. XLI patients showed significantly lower SC hydration, but normal basal trans-epidermal water loss and skin surface pH as compared to a healthy control group. Histopathology of ichthyosis epidermis showed no acanthosis, and levels of the pro-inflammatory cytokines in the corneal layer did not differ between control and lesional/non-lesional skin of XLI patients. Among the mutations in filaggrin (FLG), kallikrein 7 (KLK7), and SPINK5 genes, the prevalence of KLK7 gene mutations was significantly higher in XLI patients (50%) than in controls (0%), whereas FLG and SPINK5 prevalence was comparable. Korean XLI patients exhibited unimpaired skin barrier function and frequent association with the KLK7 gene polymorphism, which may differentiate them from Western XLI patients.
Adolescent ; Adult ; Asian Continental Ancestry Group/*genetics ; Child ; Chromosomes, Human, X ; Comparative Genomic Hybridization ; Cytokines/metabolism ; Humans ; Hydrogen-Ion Concentration ; Ichthyosis/diagnosis/*genetics/pathology ; In Situ Hybridization, Fluorescence ; Intermediate Filament Proteins/genetics ; Kallikreins/*genetics ; Male ; Polymorphism, Single Nucleotide ; Proteinase Inhibitory Proteins, Secretory/genetics ; Republic of Korea ; Skin/metabolism/*pathology ; Young Adult

X-linked ichthyosis (XLI) is a recessively inherited ichthyosis. Skin barrier function of XLI patients reported in Western countries presented minimally abnormal or normal. Here, we evaluated the skin barrier properties and a skin barrier-related gene mutation in 16 Korean XLI patients who were diagnosed by fluorescence in situ hybridization and array comparative genomic hybridization analysis. Skin barrier properties were measured, cytokine expression levels in the stratum corneum (SC) were evaluated with the tape stripped specimen from skin surface, and a genetic test was done on blood. XLI patients showed significantly lower SC hydration, but normal basal trans-epidermal water loss and skin surface pH as compared to a healthy control group. Histopathology of ichthyosis epidermis showed no acanthosis, and levels of the pro-inflammatory cytokines in the corneal layer did not differ between control and lesional/non-lesional skin of XLI patients. Among the mutations in filaggrin (FLG), kallikrein 7 (KLK7), and SPINK5 genes, the prevalence of KLK7 gene mutations was significantly higher in XLI patients (50%) than in controls (0%), whereas FLG and SPINK5 prevalence was comparable. Korean XLI patients exhibited unimpaired skin barrier function and frequent association with the KLK7 gene polymorphism, which may differentiate them from Western XLI patients.
Adolescent ; Adult ; Asian Continental Ancestry Group/*genetics ; Child ; Chromosomes, Human, X ; Comparative Genomic Hybridization ; Cytokines/metabolism ; Humans ; Hydrogen-Ion Concentration ; Ichthyosis/diagnosis/*genetics/pathology ; In Situ Hybridization, Fluorescence ; Intermediate Filament Proteins/genetics ; Kallikreins/*genetics ; Male ; Polymorphism, Single Nucleotide ; Proteinase Inhibitory Proteins, Secretory/genetics ; Republic of Korea ; Skin/metabolism/*pathology ; Young Adult

OBJECTIVETo screen the serum proteins associated with the metastasis of hepatocellular carcinoma (HCC) using a comparative proteomic approach.

METHODSThe serum samples of HCC patients with the same disease background were divided into metastatic (n=20) and non-metastatic (n=20) groups. The proteins extracted from the patients and 20 normal subjects, after depletion of the highly abundant proteins, underwent two-dimensional gel electrophoresis (2-DE). Comparative analyses of the 2-DE protein patterns between the 3 groups were conducted using a computerized image analysis system. The proteins with statistically significant differential expression between the metastatic and non-metastatic patients were identified by mass spectrometry. Western blotting was performed to examine the differential expression of the candidate proteins.

RESULTSFour protein spots were identified by mass spectrometry among the 12 differentially expressed protein spots in the serum samples of HCC patients with intrahepatic metastasis, and confirmed by searching in MASCOT database. Of the 4 proteins, cytokeratin 9 (CK9) was up-regulated by 2 folds, and inter-alpha (globulin) inhibitor H4, complement factor H-related protein 1 precursor (FHR-1), and apolipoprotein E were down-regulated by 2 folds. CK9 was found to be specifically over-expressed in the metastatic group in comparison with the non-metastatic group, as confirmed by Western blotting.

CONCLUSIONThe metastasis of HCC might be correlated to the specific variation of protein expression profiles. The overexpression of CK9 may play a crucial role in HCC metastasis, and can be used as a potential serum marker for predicting HCC metastasis.

Adult ; Blood Proteins ; genetics ; Carcinoma, Hepatocellular ; blood ; pathology ; Case-Control Studies ; Female ; Gene Expression Regulation, Neoplastic ; Glycoproteins ; blood ; genetics ; Humans ; Keratin-9 ; blood ; genetics ; Liver Neoplasms ; blood ; pathology ; Male ; Middle Aged ; Neoplasm Metastasis ; genetics ; Proteinase Inhibitory Proteins, Secretory ; blood ; genetics ; Proteomics ; Up-Regulation