The DNA damage-inducible C. elegans tankyrase is a nuclear protein closely linked to chromosomes

Liver-specific ZP domain-containing protein (LZP) was recently identified as a secreted protein that is specifically expressed in liver. However, the physiological effects of LZP are largely unknown. In this study, we found that LZP was detectable in mouse kidneys, testes, ovaries and heart, in addition to liver. LZP was localized in the spermatid cells of testes, corpus luteum cells of ovaries, and cardiac muscle cells of heart. But the protein mainly anchored on the apical membrane of the thick ascending limb of the loop of Henle (TAL) cell in mouse kidney. In rat kidney LZP and Tamm-Horsfall protein (THP) were co-localized in TAL. The in vivo interaction between LZP and THP was confirmed in kidney and urine by co-immunoprecipitation assay, and the in vitro interaction was detected by GST pull-down assay, implying that the interaction could be independent on N-linked glycosylated modification of LZP. Surprisingly, LZPs with intramolecular disulfide bridges could self-interact, and then self-aggregate into spheres of varying sizes, but not polymerize into filaments. The finding that LZP might act as a new partner of THP would provide novel insights into renal functions related to THP and LZP, such as the urothelial permeability barrier and the host defense against the adhesion of pathogens.     

LZP is required for hepatic triacylglycerol transportation through maintaining apolipoprotein B stability

The conserved zona pellucida (ZP) domain is found in hundreds of extracellular proteins that are expressed in various organs and play a variety of roles as structural components, receptors and tumor suppressors. A liver-specific zona pellucida domain-containing protein (LZP), also named OIT3, has been shown to be mainly expressed in human and mouse hepatocytes; however, the physiological function of LZP in the liver remains unclear. Here, we show that Lzp deletion inhibited very low-density lipoprotein (VLDL) secretion, leading to hepatic TG accumulation and lower serum TG levels in mice. The apolipoprotein B (apoB) levels were significantly decreased in the liver, serum, and VLDL particles of LZP-deficient mice. In the presence of LZP, which is localized to the endoplasmic reticulum (ER) and Golgi apparatus, the ER-associated degradation (ERAD) of apoB was attenuated; in contrast, in the absence of LZP, apoB was ubiquitinated by AMFR, a known E3 ubiquitin ligase specific for apoB, and was subsequently degraded, leading to lower hepatic apoB levels and inhibited VLDL secretion. Interestingly, hepatic LZP levels were elevated in mice challenged with a high-fat diet and humans with simple hepatic steatosis, suggesting that LZP contributes to the physiological regulation of hepatic TG homeostasis. In general, our data establish an essential role for LZP in hepatic TG transportation and VLDL secretion by preventing the AMFR-mediated ubiquitination and degradation of apoB and therefore provide insight into the molecular function of LZP in hepatic lipid metabolism.     

Structural conservation of mouse and rat zona pellucida glycoproteins. Probing the native rat zona pellucida proteome by mass spectrometry

The mammalian zona pellucida is an egg extracellular matrix to which sperm bind. Mouse zonae are composed of three glycoproteins (ZP1, ZP2, and ZP3), while rat zonae contain four (ZP1, ZP2, ZP3, and ZP4/ZPB). Mouse sperm bind to zonae comprised solely of mouse ZP2 and ZP3. In this report, we show that rat sperm also bind to these zonae, indicating that ZP2 and ZP3 contain a "minimum structure(s)" to which rodent sperm can bind, and ZP1 and ZP4/ZPB are dispensable in these two rodents. These data are consistent with our mass spectrometric analysis of the native rat zona pellucida proteome (defined as the fraction of the total rat proteome to which the zonae glycoproteins contribute) demonstrating that the rat zonae glycoproteins share a high degree of conservation of structural features with respect to their mouse counterparts. The primary sequences of the rat zonae proteins have been deduced from cDNA. Each zona protein undergoes extensive co- and post-translational modification prior to its secretion and incorporation into an extracellular zona matrix. Each has a predicted N-terminal signal peptide that is cleaved off once protein translation begins and an anchoring C-terminal transmembrane domain from which the mature protein is released. Mass spectrometric analysis with a limited amount of native material allowed determination of the mature N-termini of rat ZP1 and ZP3, both of which are characterized by cyclization of glutamine to pyroglutamate; the N-terminus of ZP2 was identified by Edman degradation. The mature C-termini of ZP1 and ZP3 end two amino acids upstream of a conserved dibasic residue that is part of, but distinct from, the consensus furin cleavage sequence, while the C-terminus of ZP2 was not determined. Each zona protein contains a "zona domain" with eight conserved cysteine residues that is thought to play a role in the polymerization of the zona proteins into matrix filaments. Partial disulfide bond assignment indicates that the intramolecular disulfide patterns in rat ZP1, ZP2, and ZP3 are identical to those of their corresponding mouse counterparts. Last, nearly all potential N-glycosylation sites are occupied in the rat zonae glycoproteins (three of three for ZP1, six or seven of seven for ZP2, and four or five of six for ZP3). In comparison, potential O-glycosylation sites are numerous (59-83 Ser/Thr residues), but only two regions were observed to carry O-glycans in rat ZP3.     

Human sperm do not bind to rat zonae pellucidae despite the presence of four homologous glycoproteins

The specificity of sperm-egg recognition in mammals is mediated primarily by the zona pellucida surrounding ovulated eggs. Mouse sperm are quite promiscuous and bind to human eggs, but human spermatozoa will not bind to mouse eggs. The mouse zona pellucida contains three glycoproteins, ZP1, ZP2, and ZP3, which are conserved in rat and human. The recent observation that human zonae pellucidae contain a fourth protein raises the possibility that the presence of four zona proteins will support human sperm binding. Using mass spectrometry, four proteins that are similar in size and share 62-70% amino acid identity with human ZP1, ZP2, ZP3, and ZP4/ZPB were detected in rat zonae pellucidae. However, although mouse and rat spermatozoa bind to eggs from each rodent, human sperm bind to neither, and the presence of human follicular fluid did not alter the specificity of sperm binding. In addition, mutant mouse eggs lacking hybrid/complex N-glycans or deficient in Core 2 O-glycans were no more able to support human sperm binding than normal mouse eggs. These data suggest that the presence of four zona proteins are not sufficient to support human sperm binding to rodent eggs and that additional determinants must be responsible for taxon-specific fertilization among mammals.     

Recombinant mouse ZP3 inhibits sperm binding and induces the acrosome reaction.

Mammalian fertilization involves interactions of sperm surface receptors with ligands of the zona pellucida, an extracellular matrix surrounding the ovulated egg. In mouse, the zona is composed of three glycoproteins. One of them, ZP3, participates in primary sperm binding and in the subsequent triggering of the sperm's acrosome reaction. Considerable evidence suggests that carbohydrate determinants of ZP3 are responsible for binding to sperm and may be important for acrosomal exocytosis. A full-length cDNA encoding mouse ZP3 was assembled and cloned into expression vectors that contained either a cytomegalovirus (CMV) or a vaccinia (P11) promoter. Mouse L-929 cells were stably transformed with the pZP3-CMV constructs, and green monkey CV-1 cells were infected with a recombinant vaccinia virus containing ZP3. rZP3 was affinity purified from culture media and detected on Western blots as a single 60- to 70-kDa band, which differed in molecular weight from native ZP3 (mean, 83 kDa). Nevertheless, rZP3 is biologically active. rZP3 decreases sperm-zona binding with a potency equivalent to that of native zona pellucida and, like native ZP3, rZP3 triggers acrosomal exocytosis in capacitated mouse sperm. Thus, rZP3 isolated from both rodent and primate cells appears to contain those carbohydrate and protein structures necessary for ZP3's dual role in fertilization.     

Activation of a G protein in mouse sperm by the zona pellucida,an egg-associated extracellular matrix

Mammalian sperm possess a guanine nucleotide-binding regulatory protein (G protein), with properties similar to G_i, that appears to be involved in the signal transduction pathway required for zona pellucida (ZP)-mediated acrosomal exocytosis. Mouse sperm treated with pertussis toxin (PT), a toxin that functionally inactivates G_i proteins, bind to the ZP of mouse eggs but are inhibited from undergoing acrosomal exocytosis. We have measured high-affinity GTPase activity and GTPγ[³⁵S] binding in mouse sperm homogenates incubated in the absence and presence of ZP glycoproteins isolated from either ovulated eggs or from ovarian homogenates to determine whether this extracellular matrix can activate the sperm-associated G_i protein. An increase in GTP hydrolysis (～50% over basal activity) and GTPγ[³⁵S] binding (～25–60% over basal activity) is observed when sperm homogenates are incubated in the presence of solubilized ZP glycoproteins, and the increase in GTPase activity is dependent on the concentration of ZP added to the homogenates. Accompanying this increase is a reduction in the ability of PT to catalyze in vitro [³²P]ADP-ribosylation of a M_r = 41,000 sperm G_i protein, suggesting that the increase in GTPase activity and GTPγ[³⁵S] binding is associated with the activation of a PT-sensitive sperm G protein(s). The ability of the ZP to stimulate high-affinity GTPase activity in these homogenates appears to be dependent on the capacitation state of the sperm from which the homogenates are prepared. These data suggest that a component(s) of the ZP may function in a manner similar to that of other ligands by binding to a sperm surface-associated receptor and subsequently activating a G protein coupled to an intracellular signal transduction cascade(s) required for induction of acrosomal exocytosis.     

Isolation and mapping the chicken zona pellucida genes: an insight into the evolution of orthologous genes in different species

The avian oocyte is surrounded by a specialized extracellular glycoproteinaceous matrix, the perivitelline membrane, which is equivalent to the zona pellucida (ZP) in mammals and the chorion in teleosts. A number of related ZP genes encode the proteins that make up this matrix. These proteins play an important role in the sperm/egg interaction and may be involved in speciation. The human genome is known to contain ZP1, ZP2, ZP3, and ZPB genes, while a ZPAX gene has also been identified in Xenopus. The rapid evolution of these genes has confused the nomenclature and thus orthologous relationships across species. In order to clarify these homologies, we have identified ZP1, ZP2, ZPC, ZPB, and ZPAX genes in the chicken and mapped them to chromosomes 5, 14, 10, 6, and 3, respectively, establishing conserved synteny with human and mouse. The amino acid sequences of these genes were compared to the orthologous genes in human, mouse, and Xenopus, and have given us an insight into the evolution of these genes in a variety of different species. The presence of the ZPAX gene in the chicken has highlighted a pattern of probable gene loss by deletion in mouse and gene inactivation by deletion, and base substitution in human.     

Sperm-egg recognition in the mouse: characterization of sp56, a sperm protein having specific affinity for ZP3

Recognition between mammalian gametes occurs when the plasma membrane of the sperm head binds to the zona pellucida (ZP), an extracellular coat surrounding eggs. ZP3, one of three glycoproteins in the ZP, is the egg protein recognized by sperm. A mouse sperm surface protein, sp56 (M(r) = 56,000), has been identified on the basis of its specific affinity for ZP3 (Bleil, J. D., and P. M. Wassarman. 1990. Proc. Natl. Acad. Sci. USA. 87:5563-5567). Studies presented here were designed to characterize mouse sperm sp56 and to further test whether or not this protein specifically recognizes ZP3. sp56 was purified by both ZP3 affinity chromatography and by ion exchange chromatography followed by size-exclusion chromatography. The purified native protein eluted from size-exclusion columns as a homomultimer (M(r) approximately 110,000). Each monomer of the protein contains intramolecular disulfide bonds, consistent with its extracellular location. Immunohistochemical and immunoblotting studies, using monoclonal antibodies, demonstrated that sp56 is a peripheral membrane protein located on the outer surface of the sperm head plasma membrane, precisely where sperm bind ZP3. Results of crosslinking experiments demonstrated that the ZP3 oligosaccharide recognized by sperm has specific affinity for sp56. Collectively, these results suggest that sp56 may be the sperm protein responsible for sperm-egg recognition in the mouse.     

Egg extracellular coat proteins: from fish to mammals

The extracellular coat surrounding fish (vitelline envelope; VE) and mammalian (zona pellucida; ZP) eggs is composed of long, interconnected filaments. Fish VE and mammalian ZP proteins that make up the filaments are highly conserved groups of proteins that are related to each other, as well as to their amphibian and avian egg counterparts. The rainbow trout (O. mykiss) egg VE is composed of 3 proteins, called VEalpha (approximately 58 kDa), VEbeta (approximately 54 kDa), and VEgamma (approximately 47 kDa). The mouse (M. musculus) egg ZP also is composed of 3 proteins, called ZP1 (approximately 200 kDa), ZP2 (approximately 120 kDa), and ZP3 (approximately 83 kDa). Overall, trout VE and mouse ZP proteins share approximately 25% sequence identity and have features in common; these include an N-terminal signal sequence, a ZP domain, a consensus furin cleavage-site, and a C-terminal tail. VEalpha, VEbeta, and ZP1 also have a trefoil or P-type domain upstream of the ZP domain. VEalpha and VEbeta are very similar in sequence (approximately 65% sequence identity) and are related to ZP1 and ZP2, whereas VEgamma is related to ZP3 (approximately 25% sequence identity). Mouse ZP proteins are synthesized and secreted exclusively by growing oocytes in the ovary. Trout VE proteins are synthesized by the liver under hormonal control and transported in the bloodstream to growing oocytes in the ovary. The trout VE is assembled from VEalpha/gamma and VEbeta/gamma heterodimers. The mouse ZP is assembled from ZP2/3 heterodimers and crosslinked by ZP1. Despite approximately 400 million years separating the appearance of trout and mice, and the change from external to internal fertilization and development, trout VE and mouse ZP proteins have many common structural features; as do avian and amphibian egg VE proteins. However, the site of synthesis of trout and mouse egg extracellular coat proteins has changed over time from the liver to the ovary, necessitating some changes in the C-terminal region of the polypeptides that regulates processing, secretion, and assembly of the proteins.     

Mammalian cortical granules: Contents,fate, and function

Recent studies have extended our knowledge regarding the contents of mammalian cortical granules (CG) and their function in postfertilization events. Cytochemical staining has demonstrated the presence of carbohydrates within mammalian CG, and lectin-binding studies have shown that these carbohydrates include α-Dmannose, α-D-GalNAc, and galactose residues in the hamster, α-D-mannose in the mouse and cat, and β-D-Gal(1,3)-D-GalNAc in the pig. Following fertilization and artificial activation, mannosylated material is released from CG and can be found on the oolemma and within the perivitelline space (PVS) of hamster oocytes. Fertilized or artificially activated rabbit, mouse, and human oocytes also release mannosylated, fucosylated and sialylated, and fucosylated material, respectively, which localizes to the oolemma. These glycosylated materials are probably of CG origin, although they have not been directly localized to the CG in rabbit, mice, and humans. The function(s) of the glycosylated material released from mammalian oocytes is not known, although it may participate in blocking polyspermy at the level of the plasma membrane, PVS, and/or zona pellucida (ZP), or it may facilitate preimplantation embryonic development. Proteinases, including tissue plasminogen activator, are also released from mammalian oocytes following fertilization and artificial activation, suggesting that they are of CG origin. These proteinases modify the ZP such that it is no longer receptive to sperm, and some proteinases have been suggested to bring about ZP hardening (an increased resistance to denaturing agents) by an unknown mechanism. Mouse ZP may also be hardened by an ovoperoxidase (cross-links tyrosine residues) cytochemically identified in mouse CG and CG exudate. The phenomena of ZP hardening in mammalian zygotes is not well understood but is likely to function in blocking polyspermic penetration of the ZP and/or in protecting embryos during preimplantation development. Recently, a 75 kD protein (p75) has been immunocytochemically localized to mouse CG and to the PVS of fertilized oocytes and two-cell embryos. The identity and function of p75 remains to be determined. Heparin binding placental protein may also be a CG component, since it is released from hamster oocytes following fertilization. It has not, however, been directly demonstrated to be a CG component, and its functions in fertilization and/or early embryonic development have yet to be defined. © 1994 Wiley-Liss, Inc.     

Amyloid Properties of the Mouse Egg Zona Pellucida

The zona pellucida (ZP) surrounding the oocyte is an extracellular fibrillar matrix that plays critical roles during fertilization including species-specific gamete recognition and protection from polyspermy. The mouse ZP is composed of three proteins, ZP1, ZP2, and ZP3, all of which have a ZP polymerization domain that directs protein fibril formation and assembly into the three-dimensional ZP matrix. Egg coats surrounding oocytes in nonmammalian vertebrates and in invertebrates are also fibrillar matrices and are composed of ZP domain-containing proteins suggesting the basic structure and function of the ZP/egg coat is highly conserved. However, sequence similarity between ZP domains is low across species and thus the mechanism for the conservation of ZP/egg coat structure and its function is not known. Using approaches classically used to identify amyloid including conformation-dependent antibodies and dyes, X-ray diffraction, and negative stain electron microscopy, our studies suggest the mouse ZP is a functional amyloid. Amyloids are cross-β sheet fibrillar structures that, while typically associated with neurodegenerative and prion diseases in mammals, can also carry out functional roles in normal cells without resulting pathology. An analysis of the ZP domain from mouse ZP3 and ZP3 homologs from five additional taxa using the algorithm AmylPred 2 to identify amyloidogenic sites, revealed in all taxa a remarkable conservation of regions that were predicted to form amyloid. This included a conserved amyloidogenic region that localized to a stretch of hydrophobic amino acids previously shown in mouse ZP3 to be essential for fibril assembly. Similarly, a domain in the yeast protein α-agglutinin/Sag 1p, that possesses ZP domain-like features and which is essential for mating, also had sites that were predicted to be amyloidogenic including a hydrophobic stretch that appeared analogous to the critical site in mouse ZP3. Together, these studies suggest that amyloidogenesis may be a conserved mechanism for ZP structure and function across billions of years of evolution.     

Mutation of a conserved hydrophobic patch prevents incorporation of ZP3 into the zona pellucida surrounding mouse eggs

Three glycoproteins (ZP1, ZP2, and ZP3) are synthesized in growing mouse oocytes and secreted to form an extracellular zona pellucida that mediates sperm binding and fertilization. Each has a signal peptide to direct it into a secretory pathway, a "zona" domain implicated in matrix polymerization and a transmembrane domain from which the ectodomain must be released. Using confocal microscopy and enhanced green fluorescent protein (EGFP), the intracellular trafficking of ZP3 was observed in growing mouse oocytes. Replacement of the zona domain with EGFP did not prevent secretion of ZP3, suggesting the presence of trafficking signals and a cleavage site in the carboxyl terminus. Analysis of linker-scanning mutations of a ZP3-EGFP fusion protein in transient assays and in transgenic mice identified an eight-amino-acid hydrophobic region required for secretion and incorporation into the zona pellucida. The hydrophobic patch is conserved among mouse zona proteins and lies between a potential proprotein convertase (furin) cleavage site and the transmembrane domain. The cleavage site that releases the ectodomain from the transmembrane domain was defined by mass spectrometry of native zonae pellucidae and lies N-terminal to a proprotein convertase site that is distinct from the hydrophobic patch.     

Coding sequence, genomic organization, chromosomal localization, and expression pattern of the signalosome component Cops2: the mouse homologue of Drosophila alien

The Drosophila alien gene is highly homologous to the human thyroid receptor interacting protein, TRIP15/COPS2, which is a component of the recently identified signalosome protein complex. We identified the mouse homologue of Drosophila alien through homology searches of the EST database. We found that the mouse cDNA encodes a predicted 443-amino-acid protein, which migrates at approximately 50 kDa. The gene for the mouse alien homologue, named Cops2, includes 12 coding exons spanning approximately 30 kb of genomic DNA on the central portion of mouse chromosome 2. Mouse Cops2 is widely expressed in embryonic, fetal, and adult tissues beginning as early as E7.5. Mouse Cops2 cDNA hybridizes to two mRNA bands in all tissues at approximately 2.3 and approximately 4 kb, with an additional approximately 1.9-kb band in liver. Immunostaining of native and epitope tagged proteins localized the mouse Cops2 protein in both the cytoplasm and the nucleus, with larger amounts in the nucleus in some cells.     

Structural characterization of native mouse zona pellucida proteins using mass spectrometry

The zona pellucida is an extracellular matrix consisting of three glycoproteins that surrounds mammalian eggs and mediates fertilization. The primary structures of mouse ZP1, ZP2, and ZP3 have been deduced from cDNA. Each has a predicted signal peptide and a transmembrane domain from which an ectodomain must be released. All three zona proteins undergo extensive co- and post-translational modifications important for secretion and assembly of the zona matrix. In this report, native zonae pellucidae were isolated and structural features of individual zona proteins within the mixture were determined by high resolution electrospray mass spectrometry. Complete coverage of the primary structure of native ZP3, 96% of ZP2, and 56% of ZP1, the least abundant zona protein, was obtained. Partial disulfide bond assignments were made for each zona protein, and the size of the processed, native protein was determined. The N termini of ZP1 and ZP3, but not ZP2, were blocked by cyclization of glutamine to pyroglutamate. The C termini of ZP1, ZP2, and ZP3 lie upstream of a dibasic motif, which is part of, but distinct from, a proprotein convertase cleavage site. The zona proteins are highly glycosylated and 4/4 potential N-linkage sites on ZP1, 6/6 on ZP2, and 5/6 on ZP3 are occupied. Potential O-linked carbohydrate sites are more ubiquitous, but less utilized.     

Positive effect of partial zona pellucida digestion on in vitro fertilization of mouse oocytes with cryopreserved spermatozoa

Cryopreservation of mouse spermatozoa has been widely used; however, fertility of frozen spermatozoa in some strains, especially when inseminating cryopreserved oocytes, is low and may be improved by assisted fertilization techniques. The present study was performed to investigate the effect of partial zona pellucida (ZP) digestion on the in vitro fertilization (IVF) capacity of frozen mouse spermatozoa. Mouse oocytes were subjected to partial ZP digestion using acidic Tyrode's solution (pH 3.1). Fertilization rates in digestion groups (30 or 45 s) were higher (P < 0.05) than that of zona-intact control (78.3% or 86.3% vs. 52.5%). The recovery rate at 45 s was lower (P < 0.05) than that at 30 s (84.2% vs. 97.3%). Among vitrified oocytes, the fertilization rate in treatment group (digested for 30 s) was higher (P < 0.05) than that of zona-intact group (50.8% vs. 22.1%). After embryo transfer at the two-cell stage, 17.7% and 11.8% of transferred embryos derived from fresh and vitrified digested oocytes developed to term and showed no significant difference as compared with that from zona-intact oocytes (24.1%, P > 0.05). These results indicate that partial ZP digestion improves IVF efficiency of fresh and vitrified oocytes with frozen mouse spermatozoa, which can provide valuable information for in vitro assisted fertilization using cryopreserved gametes in the re-establishment of mouse colonies.     

Identification of the true human orthologue of the mouse Zp1 gene: evidence for greater complexity in the mammalian zona pellucida?

The mammalian zona pellucida is a mixture of glycoproteins, believed to be encoded by three distinct genes, ZP1/ZPB, ZP2/ZPA, and ZP3/ZPC. We have now determined that the true human orthologue of the mouse Zp1 gene is not ZPB, but that there is a distinct human ZP1 gene. Comparison of the human ZP1 and murine Zp1 genes indicates significant conservation of nucleotide and amino acid sequences, of intron-exon size and organisation, and of regulatory sequences. In addition, the mouse and human ZP1 genes are in a region of conserved synteny between human chromosome 11 and mouse chromosome 19.     

Ovastacin, a cortical granule protease, cleaves ZP2 in the zona pellucida to prevent polyspermy

The mouse zona pellucida is composed of three glycoproteins (ZP1, ZP2, and ZP3), of which ZP2 is proteolytically cleaved after gamete fusion to prevent polyspermy. This cleavage is associated with exocytosis of cortical granules that are peripherally located subcellular organelles unique to ovulated eggs. Based on the cleavage site of ZP2, ovastacin was selected as a candidate protease. Encoded by the single-copy Astl gene, ovastacin is an oocyte-specific member of the astacin family of metalloendoproteases. Using specific antiserum, ovastacin was detected in cortical granules before, but not after, fertilization. Recombinant ovastacin cleaved ZP2 in native zonae pellucidae, documenting that ZP2 was a direct substrate of this metalloendoprotease. Female mice lacking ovastacin did not cleave ZP2 after fertilization, and mouse sperm bound as well to Astl-null two-cell embryos as they did to normal eggs. Ovastacin is a pioneer component of mouse cortical granules and plays a definitive role in the postfertilization block to sperm binding that ensures monospermic fertilization and successful development.     

Identification and characterization of D8C, a novel domain present in liver-specific LZP, uromodulin and glycoprotein 2, mutated in familial juvenile hyperuricaemic nephropathy

Present work reported a novel domain--D8C (domain with conserved eight cysteines in liver-specific ZP domain-containing protein, glycoprotein 2 (GP-2) and uromodulin (UMOD)), present in liver-specific LZP, UMOD, GP-2 and some uncharacterized proteins, most of which are membrane proteins, extracellular proteins or nuclear membrane proteins. D8C contains eight well-conserved cysteine residues, which were predicted to form four pairs of disulfide bridges. D8C is composed mainly of beta-strands. Mutation in the D8C at Cys217 in human UMOD is associated with familial juvenile hyperuricaemic nephropathy, which might be due to the disruption of the disulfide bridge. Identification of D8C would further the understandings of related proteins.