Midori Nomura

Disruption of Mouse CD46 Causes an Accelerated Spontaneous Acrosome Reaction in Sperm

ABSTRACT Human membrane cofactor protein (MCP, CD46) is a ubiquitously expressed protein known to protect cells from complement attack. Interestingly, when we examined the expression of mouse CD46, which we recently cloned, the message was found only in testis and the protein was found on the inner acrosomal membrane of sperm. In order to elucidate the function of CD46, we produced mice carrying a null mutation in the CD46 gene by using homologous recombination. Despite the absence of CD46, the mice were healthy and both sexes were fertile. However, to our surprise, the fertilizing ability of males appeared to be facilitated by disruption of the CD46 gene, as the average number of pups born from CD46−/− males was significantly greater than that of wild-type males. It was also revealed that the incidence of the spontaneous acrosome reaction doubled in CD46−/− sperm compared to that in wild-type sperm. It was assumed that this increase caused the heightened fertilizing ability found in CD46−/− sperm. These data suggest that CD46 may have some role in regulating sperm acrosome reaction.

Human membrane cofactor protein (MCP, CD46): multiple isoforms and functions.

Human membrane cofactor protein (MCP, CD46) is a 45-70 kDa protein with genetic and tissue-specific heterogeneity, and is expressed on all nucleated cells. MCP consists from N-terminus of 4 short consensus repeats (SCRs), 1-3 serine/threonine-rich (ST) domains, a transmembrane domain (TM) and a cytoplasmic tail (CYT). More than 8 isoforms are generated secondary to alternative splicing due to combinations of various exons encoding the ST, TM and CYT domains. It serves as a cofactor of serine protease factor I for inactivation of complement C3b and C4b. Its primary role is to protect host cells from homologous complement attack by inactivating C3b/C4b deposited on the membrane. It also acts as receptors for measles virus (MV), some kinds of bacteria and for a putative ligand on oocytes. MV infection causes temporal host immune suppression, which may appear secondary to signaling events through MCP on macrophages and dendritic cells. These functional properties of human MCP may facilitate xenotransplantation and may be useful in the generation of animal models of measles by creating human MCP-expressing animals.

A Novel Chicken Membrane-Associated Complement Regulatory Protein: Molecular Cloning and Functional Characterization

A cDNA encoding a membrane-associated complement (C) regulatory protein was identified here for the first time in an oviparous vertebrate, chicken. This protein, named Cremp, possessed five short consensus repeats (SCRs) and one SCR-like domain followed by a transmembrane domain and a cytoplasmic tail. SCR1/SCR2 of Cremp were 43.6% identical with SCR2/SCR3 of human decay-accelerating factor (CD55), and SCR3/SCR4 were 45.3% identical with those of human membrane cofactor protein (CD46). Cremp is likely to be an ancestral hybrid protein of human decay-accelerating factor and membrane cofactor protein rather than a homolog of rodent C receptor 1-related protein y, which structurally resembles human CR1 (CD35). Chinese hamster ovary cells transfected with Cremp were efficiently protected from chicken C but not from human or rabbit C in both classical and alternative pathways. Thus, chicken Cremp is a membrane C regulator for cell protection against homologous C. Cremp mRNA was seen as a doublet comprised of a faint band of 2.2 kb and a thick band of 3.0 kb on RNA blotting analysis. An Ab against chicken Cremp recognized a single band of 46.8 kDa on immunoblotting. mRNA and protein of Cremp were ubiquitously expressed in all chicken organs tested. Minute amounts of dimer were present in some tissues. Surface expression of Cremp was confirmed by flow cytometry and immunofluorescence analysis. These results suggested that even in nonmammals a C regulatory membrane protein with ubiquitous tissue distribution should be a prerequisite for protection of host cells from homologous C attack.

Genomic analysis of idiopathic infertile patients with sperm-specific depletion of CD46.

Three infertile subjects with no expression of CD46 (membrane cofactor protein of complement) on their spermatozoa were found when screening 542 idiopathic male infertile patients. The sperm CD46 isoform was reported to be associated with the sperm-egg interaction, yet a ubiquitous expression of CD46 confers resistance to complement-mediated injury on host cells. All three patients expressed normal CD46 isoforms on their lymphocytes and granulocytes. Thus, the loss of CD46 is sperm-specific, probably due to testicular germ cell-specific regulation of CD46 production. Recently, a mechanism of the gene regulation of human CD46 was elucidated in which the silencer element of the 3′ UT and the promoter region of human CD46 gene partly participate. Here, we analyzed these regions of the CD46 gene in our 3 patients. We found no abnormality in 3′ and 5′ regions of the CD46 genome in the 3 patients. Thus, in these infertile patients sperm-specific depletion of CD46 is not governed by the so for identified regulators in the CD46 gene. Other unknown factors outside the known regulatory regions would play a role in the regulation of sperm-specific CD46 expression.

Mechanism of host cell protection from complement in murine cytomegalovirus (CMV) infection: identification of a CMV‐responsive element in the CD46 promoter region

Mouse cells ubiquitously express CRRY, which is a functional orthologue of human decay‐accelerating factor (DAF; CD55) and membrane cofactor protein (MCP; CD46), and thus protects cells from homologous complement. NIH3T3 cells expressed minute levels of mouse CD46 (mCD46) mRNA but barely produced mCD46 protein. mCD46 message and protein levels were markedly increased during mouse cytomegalovirus (mCMV) infection. Consistently, mCD46‐expressing cells became resistant to mouse complement; primary‐cultured fibroblasts from mCD46 gene‐disrupted mice showed no increase in protection, resulting in complement‐dependent cytolysis. Thus, the marked up‐regulation of mCD46 in mouse fibroblast cells/cell lines by mCMV infection participates in host cell protection from complement. By mCD46 promoter deletion assay, the region necessary for induction of the promoter activity by mCMV infection was shown to be restricted to a sequence of 19 bp, which was homologous to the correspondingportion in human CD46, and the promoter regions of early‐inducible human CMV UL36 and human herpesvirus 6 UL29. The results were confirmed by mutation analysis of this 19‐bp region. We designated this sequence as the CMV‐responsive element (CMVRE). Electrophoretic mobility shift assay demonstrated the existence of a CMVRE‐binding factor, expression of which was significantly increased after mCMV infection. Thus, mCMV up‐regulates the gene expression of mCD46 via CMVRE and CMVRE‐binding factor, resulting in mCD46 protein expression on mCMV‐infected cells. Since both the membrane and soluble mCD46retained complement regulatory activity, mCD46 induced by mCMV infection may act as a regulator of systemic complement activation. This represents a unique strategy of mCMV survival in host cells with sufficient replication by circumventing host complement attack.

A Comparative Analysis of the Antigenic, Structural, and Functional Properties of Three Different Preparations of Recombinant Human Interleukin-18

We compared the structural and functional properties of three recombinant human interleukin-18 (rIL-18) preparations, commercially available (Pep rIL-18) and prepared in our laboratory (active and inactive, according to their ability to potentiate IL-12-mediated interferon-gamma [IFN-gamma] induction in lymphocytes). All three preparations showed multimer formation on SDS-PAGE/immunoblotting using monoclonal antibodies (mAb) against the inactive form of rIL-18. In contrast, only the 18-kDa bands were recognized in each sample by mAb against the active form of rIL-18. The amounts of multimers and the 18-kDa moiety of Pep rIL-18 resembled those of the inactive rather than the active form. Likewise, the reaction profile of Pep rIL-18 toward mAb was very similar to that of inactive but not active rIL-18 on sandwich ELISA. Pep rIL-18 potentiated IFN-gamma-inducing activity together with IL-12, but its potency was 100-fold less than that of the active rIL-18, and excess doses were required for its activity. The inactive rIL-18 showed virtually no IFN-gamma-inducing ability, but when reduced and reconstituted, it inhibited the IFN-gamma-inducing activity of active rIL-18. These results suggest that there are two categories of recombinant IL-18 that are structurally, functionally, and antigenically different, and the mAb 125-2H and 21 can discriminate these two IL-18 populations by recognizing the epitopes specifically expressed on active and inactive IL-18, respectively.

One of the retinoic acid‐inducible cDNA clones in mouse embryonal carcinoma F9 cells encodes a novel isoenzyme of fructose 1,6‐bisphosphatase

Rae‐30, one of the retinoic acid (RA)‐inducible cDNA clones in mouse embryonal carcinoma F9 cells, was sequenced and the deduced RAE‐30 protein showed about a 70% homology to mammalian fructose 1,6‐bisphosphatase (EC 3.1.3.11) (FBPase), in comparison to over 85% homology observed among the previously documented rat liver, pig kidney and human leukemic HL‐60 cell FBPases. The Rae‐30 mRNAs were not detected in various tissues of adult mice, including the liver and kidney, but were detected in a placenta and predominantly in the intestine of adult mice. These findings indicate that the Rae‐30 cDNA encodes a novel isoenzyme of FBPase, which is likely to be involved in early differentiation in mammalian cells.

Innate Immune Therapy For Cancer

Tumor cells usually express antigens which are distinguishable from normal “self” antigens and are thereby recognized by the host immune system. However, the host immune system barely responds to tumors in patients. Supplementation with adjuvant (such as BCG-CWS) in patients with cancer contributes to regression of intrinsically growing cancer. The adjuvant targets antigen-presenting cells, i.e. innate immunity, but not lymphocytes, and promotes up-regulation of MHC, co-stimulators and initial cytokines in antigen-presenting cells. We hypothesized that the role of the adjuvant is to provide conditions suitable for antigen-presentation where antigens are available and the lack of adjuvant-induced priming of antigen-presenting cells results in unresponsiveness to tumor antigens. Here, we report innate immune therapy applicable to cancer patients by supplementation with adjuvants for induction of potent immune responses against tumors.

Isolation of a cDNA clone encoding mouse 3-hydroxyacyl CoA dehydrogenase ☆

Rae-38, a cDNA clone isolated from mouse embryonal carcinoma F9 cells, was sequenced, and the deduced RAE-38 protein showed about 86% homology to pig 3-hydroxyacyl CoA dehydrogenase (HCDH; EC 1.1.1.35). This clone can be used to elucidate the regulatory mechanism of HCDH gene expression in mammals.

THE 3'-UT OF THE UBIQUITOUS MRNA OF HUMAN CD46 CONFERS SELECTIVE SUPPRESSION OF PROTEIN PRODUCTION IN MURINE CELLS

Mice express CD46 protein and its approximately 1.5‐kb mRNA only in the testicular germ cells, unlike primates and pigs which ubiquitously express CD46 and its approximately 4 kb mRNA. Human CD46 is not well expressed in transgenic mice carrying human CD46 cDNA. To analyze the mechanism of regulation of human CD46 expression in mouse cells, we cloned the long (ubiquitous ∼ 4 kb, L‐form) and short (∼ 1.5 kb, S‐form) forms of human CD46 cDNA whose size difference is due to a stretch of the 3′‐UT. Transfection of either cDNA resulted in marked S‐form‐dependent protein generation in all mouse cell lines tested. In contrast, there were virtually no differences in protein synthesis between S‐ and L‐form cDNA in the simian and swine cell lines. Quantitative mRNA analyses and luciferase reporter gene assays suggested that one major cause of this interspecies discrepancy is transcriptional regulation, i.  e. selective suppression of the 4‐kb mRNA leading to low levels of protein synthesis. Although other mechanisms such as mRNA stability and translational regulation may lead to the low expression levels of L‐form‐derived CD46 in mice, the silencer activity in the L‐form 3′‐UT appears to function in human CD46 transcriptional regulation in mice.