Katsuya Kominami

Green mice' as a source of ubiquitous green cells

The green fluorescent protein (GFP) is responsible for the green bioluminescence of the jellyfish Aequorea victoria. Many classes of GFP mutants exist that display modified fluorescence spectra and an increased extinction coefficient. We produced transgenic mouse lines with an ‘enhanced’ GFP (EGFP) cDNA under the control of a chicken beta‐actin promoter and cytomegalovirus enhancer. All of the tissues from these transgenic lines, with the exception of erythrocytes and hair, were green under excitation light. The fluorescent nature of the cells from these transgenic mouse lines would facilitate their use in many kinds of cell transplantation experiments.

The putative chaperone calmegin is required for sperm fertility

The proper folding of newly synthesized membrane proteins in the endoplasmic reticulum (ER) is required for the formation of functional mature proteins. Calnexin is a ubiquitous ER chaperone that plays a major role in quality control by retaining incompletely folded or misfolded proteins. In contrast to other known chaperones such as heat-shock proteins, BiP and calreticulin, calnexin is an integral membrane protein. Calmegin is a testis-specific ER protein that is homologous to calnexin. Here we show that calmegin binds to nascent polypeptides during spermatogenesis, and have analysed its physiological function by targeted disruption of its gene. Homozygous-null male mice are nearly sterile even though spermatogenesis is morphologically normal and mating is normal. In vitro, sperm from homozygous-null males do not adhere to the egg extracellular matrix (zona pellucida), and this defect may explain the observed infertility. These results suggest that calmegin functions as a chaperone for one or more sperm surface proteins that mediate the interactions between sperm and egg. The defective zona pellucida-adhesion phenotype of sperm from calmegin-deficient mice is reminiscent of certain cases of unexplained infertility in human males.

A rapid and non‐invasive selection of transgenic embryos before implantation using green fluorescent protein (GFP)

Non‐invasive selection of transgenic mice was performed at the stage of preimplantation embryos. The morulae collected from wild female mated with hemizygous transgenic male expressing Aequorea victoria green fluorescent protein (GFP) under chicken β‐actin promoter could be classified as green or non‐green under a fluorescent microscope. All the green embryos were shown to carry the transgene by PCR analysis. Taking advantage of the detection of GFP expression can be done non‐invasively, the selected embryos were demonstrated to be able to developed to term with 100% of accuracy of the selection.

CESSATION OF SPERMATOGENESIS IN JUVENILE SPERMATOGONIAL DEPLETION (JSD/JSD) MICE

Background:

Characterization of a brain-related serine protease, neurosin (human kaillikrein 6), in human cerebrospinal fluid.

Neurosin (also known as zyme or protease M) is a trypsin-like serine protease dominantly expressed in the human brain. According to the official nomenclature, this gene is now designated as human kallikrein 6 (KLK6) and the protein is designated hK6. To investigate the metabolism of neurosin in human brain, neurosin contained in the human cerebrospinal fluid (CSF) was analyzed. Neurosin was detected in the all CSFs tested by Western blot analysis using an anti-neurosin monoclonal antibody. We purified neurosin from CSF (CSF-neurosin) using an immunoaffinity chromatography and an anion-exchange chromatography. SDS-PAGE revealed that the purified protein has a relative mol. mass (Mr) of 25,000 Da. The observed sequence of the N-terminal amino acids, Glu-Glu-Gln-Asn-Lys, of the purified CSF-neurosin was identical to the sequence of N-terminal of the pro-enzyme form, which is presumed to have no enzyme activity. CSF-neurosin neither showed any enzyme activity to Boc-Phe-Ser-Arg-4-methylcoumaryl-7-amide, which is known to be degraded by the mature neurosin, nor cleaved gelatin. To confirm that the major portion of CSF-neurosin is present in the pro-enzyme form, Western blot analysis using antibodies specific to the pro- or mature enzyme was carried out. The antibody against the mature neurosin fragment did not react with CSF-neurosin. Only the antibody against the pro-enzyme fragment detected CSF-neurosin. Thus, our results suggest that neurosin is present as an inactive pro-enzyme in the human CSF.

cDNA cloning and tissue-specific splicing variants of mouse hippostasin/TLSP (PRSS20)

Two splicing variants of mouse hippostasin/TLSP (PRSS20) were identified and termed brain-type and prostate-type, respectively. Mouse hippostasin/TLSP showed 76.8% identity to the human homologue. Transient expression showed that both translational products were secreted into the conditioned medium. Mouse hippostasin/TLSP was expressed preferentially in the fetal brain and the prostate, but not in the neonatal brain. The brain expressed only brain-type hippostasin/TLSP, while the prostate expressed both types.

Multiple promoters regulate tissue‐specific alternative splicing of the human kallikrein gene, KLK11/hippostasin

The human kallikrein (KLK) family consists of 15 genes located on human chromosome 19q13.4. KLK11/hippostasinis a member of the kallikrein family and is expressed in various tissues. Two types of KLK11 isoforms, isoform 1 and isoform 2, have been predicted from cDNA sequences. Isoform 1 has been isolated from human hippocampus, whereas isoform 2 has been isolated from prostate. However, the regulation and characteristics of these isoforms are unknown. We identified the first three exons (1a, 1b, and 1c) by determining their transcription initiation sites. Exon 1b contained the initiation codon of isoform 2, and noncoding exons 1a and 1c contributed to isoform 1 mRNA. The dual luciferase promoter assay revealed three promoter regions, corresponding to the first exon of each isoform. Reverse transcription and PCR showed that exon 1a was expressed in the hippocampus, thalamus, and non‐central nervous system (CNS) tissues, whereas exon 1b was detected only in non‐CNS tissues. Exon 1c was observed in both CNS and non‐CNS tissues, except for salivary glands. In vitro mutagenesis revealed that the initiation codon for isoform 2 in exon 1b was functional. Isoform 2 had additional hydrophilic amino acids at the amino terminal and was secreted from the neuroblastoma cell line Neuro2a. Isoform 1 fused with green fluorescent protein (GFP) was distributed to cellular processes, whereas isoform 2–GFP was retained in the Golgi apparatus. We suggest that not only alternative splicing but also tissue‐specific use of multiple promoters regulate the expression and intracellular trafficking of KLK11/hippostasin isoforms.

A novel serine protease highly expressed in the pancreas is expressed in various kinds of cancer cells

We have isolated a cDNA that encodes a novel serine protease, prosemin, from human brain. The cDNA of human prosemin is 1306 bp, encoding 317 amino acids. It showed significant homology with the sequence of a chromosome 16 cosmid clone (accession no. NT_037887.4). The prosemin gene contains six exons and five introns. The amino acid sequence of prosemin shows significant homology to prostasin, γ‐tryptase, and testisin (43%, 41%, and 38% identity, respectively), the genes of which are also located on chromosome 16. Northern hybridization showed that prosemin is expressed predominantly in the pancreas and weakly in the prostate and cerebellum. However, western blot and RT‐PCR analyses showed that prosemin is expressed and secreted from various kinds of cancer cells, such as glioma, pancreas, prostate, and ovarian cell lines. Prosemin is secreted in the cystic fluid of clinical ovarian cancers. Furthermore, immunohistochemistry showed prosemin protein localized in the apical parts of ovarian carcinomas. Recombinant prosemin was expressed in COS cells and was purified by immunoaffinity chromatography. Recombinant prosemin preferentially cleaved benzyloxycarbonyl (Z)‐His‐Glu‐Lys‐methylcoumaryl amidide (MCA) and t‐butyloxycarbonyl (Boc)‐Gln‐Ala‐Arg‐MCA. Our results suggest that prosemin is a novel serine protease of the chromosome 16 cluster that is highly expressed in the pancreas. The usefulness of this serine protease as a candidate tumor marker should be further examined.