Uterus-specific transcriptional regulation underlies eggshell pigment production in Japanese quail.

The precursor of heme, protoporphyrin IX (PPIX), accumulates abundantly in the uteri of birds, such as Japanese quail, Coturnix japonica, which has brown-speckled eggshells; however, the molecular basis of PPIX production in the uterus remains largely unknown. Here, we investigated the cause of low PPIX production in a classical Japanese quail mutant exhibiting white eggshells by comparing its gene expression in the uterus with that of the wild type using transcriptome analysis. We also performed genetic linkage analysis to identify the causative genomic region of the white eggshell phenotype. We found that 11 genes, including 5'-aminolevulinate synthase 1 (ALAS1) and hephaestin-like 1 (HEPHL1), were specifically upregulated in the wild-type uterus and downregulated in the mutant. We mapped the 172 kb candidate genomic region on chromosome 6, which contains several genes, including a part of the paired-like homeodomain 3 (PITX3), which encodes a transcription factor. ALAS1, HEPHL1, and PITX3 were expressed in the apical cells of the luminal epithelium and lamina propria cells of the uterine mucosa of the wild-type quail, while their expression levels were downregulated in the cells of the mutant quail. Biochemical analysis using uterine homogenates indicated that the restricted availability of 5'-aminolevulinic acid is the main cause of low PPIX production. These results suggest that uterus-specific transcriptional regulation of heme-biosynthesis-related genes is an evolutionarily acquired mechanism of eggshell pigment production in Japanese quail. Based on these findings, we discussed the molecular basis of PPIX production in the uteri of Japanese quails.

Dual enzymatic properties of the cytoplasmic peptide: N-glycanase in C. elegans.

The endoplasmic reticulum-associated degradation (ERAD) of misfolded (glyco)proteins ensures that only functional, correctly folded proteins exit from the ER and that misfolded ones are degraded by the ubiquitin-proteasome system. During the degradation of misfolded glycoproteins, some of them are subjected to deglycosylation by the cytoplasmic peptide:N-glycanase (PNGase). The cytosolic PNGase is widely distributed throughout eukaryotes. Here we show that the nematode Caenorhabditis elegans PNG-1, the cytoplasmic PNGase orthologue in this organism, exhibits dual enzyme functions, not only as PNGase but also as an oxidoreductase (thioredoxin). Using an in vitro assay as well as an in vivo assay system in budding yeast, the N-terminal thioredoxin domain and the central transglutaminase domain were found to be essential for oxidoreductase activity and PNGase activity, respectively. Occurrence of a C. elegans mutation affecting a catalytic residue in the PNGase domain strongly suggests the functional importance of this protein in higher eukaryotes.

Man2C1, an alpha-mannosidase, is involved in the trimming of free oligosaccharides in the cytosol.

The endoplasmic-reticulum-associated degradation of misfolded (glyco)proteins ensures that only functional, correctly folded proteins exit from the endoplasmic reticulum and that misfolded ones are degraded by the ubiquitin-proteasome system. During the degradation of misfolded glycoproteins, they are deglycosylated by the PNGase (peptide:N-glycanase). The free oligosaccharides released by PNGase are known to be further catabolized by a cytosolic alpha-mannosidase, although the gene encoding this enzyme has not been identified unequivocally. The findings in the present study demonstrate that an alpha-mannosidase, Man2C1, is involved in the processing of free oligosaccharides that are formed in the cytosol. When the human Man2C1 orthologue was expressed in HEK-293 cells, most of the enzyme was localized in the cytosol. Its activity was enhanced by Co2+, typical of other known cytosolic alpha-mannosidases so far characterized from animal cells. The down-regulation of Man2C1 activity by a small interfering RNA drastically changed the amount and structure of oligosaccharides accumulating in the cytosol, demonstrating that Man2C1 indeed is involved in free oligosaccharide processing in the cytosol. The oligosaccharide processing in the cytosol by PNGase, endo-beta-N-acetylglucosaminidase and alpha-mannosidase may represent the common 'non-lysosomal' catabolic pathway for N-glycans in animal cells, although the molecular mechanism as well as the functional importance of such processes remains to be determined.

Site-specific labeling of cytoplasmic peptide:N-glycanase by N,N'-diacetylchitobiose-related compounds.

Peptide:N-glycanase (PNGase) is the deglycosylating enzyme, which releases N-linked glycan chains from N-linked glycopeptides and glycoproteins. Recent studies have revealed that the cytoplasmic PNGase is involved in the degradation of misfolded/unassembled glycoproteins. This enzyme has a Cys, His, and Asp catalytic triad, which is required for its enzymatic activity and can be inhibited by "free" N-linked glycans. These observations prompted us to investigate the possible use of haloacetamidyl derivatives of N-glycans as potent inhibitors and labeling reagents of this enzyme. Using a cytoplasmic PNGase from budding yeast (Png1), Man9GlcNAc2-iodoacetoamide was shown to be a strong inhibitor of this enzyme. The inhibition was found to be through covalent binding of the carbohydrate to a single Cys residue on Png1, and the binding was highly selective. The mutant enzyme in which Cys191 of the catalytic triad was changed to Ala did not bind to the carbohydrate probe, suggesting that the catalytic Cys is the binding site for this compound. Precise determination of the carbohydrate attachment site by mass spectrometry clearly identified Cys191 as the site of covalent attachment. Molecular modeling of N,N'-diacetylchitobiose (chitobiose) binding to the protein suggests that the carbohydrate binding site is distinct from but adjacent to that of Z-VAD-fmk, a peptide-based inhibitor of this enzyme. These results suggest that cytoplasmic PNGase has a separate binding site for chitobiose and other carbohydrates, and haloacetamide derivatives can irreversibly inhibit that catalytic Cys in a highly specific manner.

Complexes of gelatinases and tissue inhibitor of metalloproteinases in human seminal plasma.

Previously reported data have indicated the existence of two kinds of matrix metalloproteinases (MMP-2 and MMP-9) in human seminal plasma (Shimokawa et al, 2002). Here we report the existence of complexes of gelatinases and tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2 in human seminal plasma. After the seminal plasma supernatant was separated on a gel-filtration column chromatography of GCL-2000-sf-cellulofine. Western blot analysis showed these proteins were recognized by two antibodies to TIMP-1 and TIMP-2, but not to TIMP-3 or TIMP-4. These bands were consistent with standard recombinant full-length TIMP-1 and TIMP-2 proteins. These bands had molecular weights of approximately 29 and 21 kd for TIMP-1 and TIMP-2, respectively. These proteins existed as complexes of proMMP-9/TIMP-1, proMMP-2/TIMP-2, MMP-2/TIMP-2, free TIMP-1, and TIMP-2 in human seminal plasma. The partially free TIMPs were degradated by some proteinases in human seminal plasma. These results indicate two kinds of TIMPs (TIMP-1 and TIMP-2) and their complexes with progelatinases in human seminal plasma.

Identification of complexes of gelatinase A and tissue inhibitor of metalloproteinase-2 in human follicular fluid.

Background and Aims: Ovulation involves considerable tissue remodeling in normal ovarian function. These processes are expected to involve matrix metalloproteinases (MMP). Follicular rupture is caused by the degradation of the basement membrane between the thecal and granulose layers, as well as disruption of the extracellular matrix (ECM) at the site of rupture. We report on the existence of the complexes of progelatinase A (proMMP-2), MMP-2 and a tissue inhibitor of metalloproteinase-2 (TIMP-2) using zymographic and immunological techniques in human follicular fluid (HFF). Methods and Results: Partial purification of the complexes was achieved by using gelatin affinity column chromatography. The peak (tubes 68-73) in this chromatography showed gelatinase activities by gelatin-zymography, and also an inhibition by EDTA (metalloproteinase inhibitor). The molecular weights of the gelatinase activities were approximately 72 and 67 kDa, and were consistent with standard proMMP-2 and MMP-2, as found by using gelatin-zymography. Similarly, the band in this peak was consistent with standard recombinant full-length TIMP-2, as found by the use of western blot analysis, and the molecular weight of the band was approximately 21 kDa. Conclusion: As proMMP-2, MMP-2 and TIMP-2 exist in the peak from the gelatin affinity column, we expected that these form the complexes. These results indicate that the complexes of proMMP-2/TIMP-2 and MMP-2/TIMP-2 exist in HFF. (Reprod Med Biol 2003; 2: 115-119).

Matrix metalloproteinase (MMP)-2 and MMP-9 activities in human seminal plasma.

We report on the existence of two kinds of matrix metalloproteinases (MMPs), MMP-2 and MMP-9, in human seminal plasma. Partial purification of the proteinases was achieved by two steps, consisting of chromatography on a gel-filtration column and then on a gelatin affinity column. Proteinase activities in the chromatography extracts were shown to hydrolyse a fluorescent substrate specific to MMPs (Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2). The proteinases were detected using gelatin-zymography, but were not detected using casein-zymography, and were also inhibited by EDTA, EGTA and o-phenanthroline. Molecular weights of the proteinases were determined by SDS-PAGE, gelatin-zymography and Western blot to be approximately 92, 84, 72, 67, 52 and 45 kDa. Gelatin-zymography showed three major bands of activity at 72, 67 and 52 kDa and minor bands at 92, 84 and 45 kDa. Apart from the two smallest bands, these proteinases were all recognized by the polyclonal antibodies for MMP-2 or MMP-9. These results indicate that two kinds of pro-form and active-form matrix metalloproteinases, MMP-2 and MMP-9, and their degradation products, are present in human seminal plasma.