Molecular cloning of complementary DNA encoding mouse seminal vesicle-secreted protein SVS I and demonstration of homology with copper amine oxidases

The primary structure of mouse SVS I was determined by peptide sequencing and nucleotide sequencing of cloned cDNA. The precursor molecule consists of 820 amino acid residues, including a signal peptide of 24 residues, and the mature polypeptide chain of 91 kDa has one site for potential N-linked glycosylation. The SVS I is homologous with amiloride-binding protein 1 (ABP1), a diamine oxidase. However, it probably lacks enzymatic activity, because the cDNA codes for His instead of Tyr at the position of the active-site topaquinon. The SVS I monomer probably binds one molecule of copper, because the His residues coordinated by Cu(II) are conserved. The SVS I gene consists of five exons and is situated on mouse chromosome 6,B2.3. It is located in a region of 100 kilobases (kb) containing several genes with homology to SVS I, including the gene of ABP1 and two other proteins with homology to diamine oxidase. The locus is conserved on rat chromosome 4q24, but the homologous region on human chromosome 7q34-q36 solely contains ABP1. The other genes with homology to diamine oxidase were probably present in a progenitor of primates and rodents but were lost in the evolutionary lineage leading to humans-presumably during recombination between chromosomes. The estimated molecular mass of rat SVS I is 102 kDa (excluding glycosylation). The species difference in size of SVS I is caused by tandem repeats of 18 amino acid residues in the central part of the molecule: The mouse has seven repeats, and the rat has 12 repeats.     

Cysteine residue periodicity is a conserved structural feature of variable surface proteins from Paramecium tetraurelia.

The DNA sequences of the entire coding regions of the A and C type variable surface protein genes from Paramecium tetraurelia, stock 51 have been determined. The 8151 nucleotide open reading frame of the A gene contains several tandem repeats of 210 nucleotides within the central portion of the molecule as well as a periodic structure defined by cysteine residues. The 6699 nucleotide open reading frame of the C gene does not contain any identifiable tandem repeats or internal similarity but maintains a periodicity based on the cysteine residue spacing. The deduced amino acid sequences encoded by the two genes are most similar within the 600 amino-terminal and 600 carboxyl-terminal amino acid residues, the central portions show only limited sequence similarity. We conclude that internal repeats are not a conserved feature of variable surface proteins in Paramecium and discuss the possible importance of the regular pattern of cysteine residues.     

A novel heat-labile phospholipid-binding protein, SVS VII, in mouse seminal vesicle as a sperm motility enhancer

SVS VII, one of seven major proteins in mouse seminal vesicle secretion, was purified to homogeneity. Neither glycoconjugate nor free thiol group was detected in the protein. The primary structure deduced from the corresponding cDNA was confirmed using amino acid sequence determination, which supported the finding that SVS VII consists of 76 amino acid residues with five disulfide bridges. Accordingly, it has a theoretical molecular mass of 8538, which was proven using the mass spectrum of SVS VII. The CD spectrum of SVS VII in 50 mm phosphate buffer at pH 7.4 appeared as one negative band arising from the beta form at 217 nm and several fine structures due to nonpeptide chromophores including a prominent band for the disulfide bond at 250 nm. This, together with the predicted secondary structures, indicated no helices but a mixture of beta form, beta turn, and unordered form in SVS VII. A cytochemical study illustrated the presence of the SVS VII-binding region on the entire surface of mouse sperm. The SVS VII-sperm binding was inhibited by the dispersed sperm lipids. The results of TLC overlay assay for the binding of (125)I-SVS VII to phospholipids and the interaction between SVS VII and phospholipid liposomes demonstrated a specific binding of this protein to both phosphatidylethanolamine and phosphatidylserine. The SVS VII-sperm binding greatly enhanced sperm motility but did not induce sperm capacitation. Heating the protein solution for 10 min at 90 degrees C unfolded the protein molecule, and the unfolded SVS VII immobilized the sperm.     

Identification of the major TG4 cross‐linking sites in the androgen‐dependent SVS I exclusively expressed in mouse seminal vesicle

SVS I was exclusively expressed in seminal vesicle in which the protein was immunolocalized primarily to the luminal epithelium of mucosal folds. The developmental profile of its mRNA expression was shown to be androgen‐dependent, manifesting a positive correlation with the animal's maturation. There are 43 glutamine and 43 lysine residues in one molecule of SVS I, which is one of the seven major monomer proteins tentatively assigned on reducing SDS–PAGE during the resolution of mouse seminal vesicle secretion. Based on the fact that SVS I‐deduced protein sequence consists of 796 amino acid residues, we produced 7 recombinant polypeptide fragments including residues 1–78/F1, residues 79–259/F2, residues 260–405/F3, residues 406–500/F4, residues 501–650/F5, residues 651–715/F6, and residues 716–796/F7, and measured the covalent incorporation of 5‐(biotinamido)pentylamine (BPNH₂) or biotin‐TVQQEL (A25 peptide) to each of F1‐to‐F7 by type 4 transglutaminase (TG₄) from the coagulating gland secretion. F2 was active to a greater extent than the other fragments during the BPNH₂‐glutamine incorporation, and a relatively low extent of A25‐lysine cross link was observed with all of the seven fragments. The MS analysis of BPNH₂‐F2 conjugate identified Q²³² and Q²⁵⁴ as the two major TG₄ cross‐linking sites. This was substantiated by the result that much less BPNH₂ was cross‐linked to any one of the three F2 mutants, including Q232G and Q254G obtained from single‐site mutation, and Q232G/Q254G from double‐site mutation. J. Cell. Biochem. 107: 899–907, 2009. © 2009 Wiley‐Liss, Inc.     

Homology between rabbit uteroglobin and the rat seminal vesicle sperm-binding protein: Prediction of structural features of glutamine substrates for transglutaminase

A comparison of both amino acid composition and sequence of the rabbit uteroglobin (UG) subunit and the rat seminal vesicle sperm-binding protein (rSBP) by computer analysis indicates homology between the two polypeptide chains. These findings are supported by immunological studies showing the occurrence of similar antigenic determinants. In addition, our data indicate the glutamine-9 of the rat seminal vesicle sperm-binding protein and glutamine-40 of UG as the possible glutamine residues involved when the proteins act as transglutaminase (TGase) substrates. The latter results represent an interesting approach to determining the general structural features of the acyl donor site in the TGase-catalyzed reaction.     

Mutual adaptation between mouse transglutaminase 4 and its native substrates in the formation of copulatory plug

Formation of copulatory plugs by male animals is a common means of reducing competition with rival males. In mice, copulatory plugs are formed by the coagulation of seminal vesicle secretion (SVS), which is a very viscous and self-clotting fluid containing high concentration of proteins. In its native state, mouse SVS contains a variety of disulfide-linked high-molecular-weight complexes (HMWCs) composed of mouse SVS I-III, which are the major components of mouse SVS. Further, mouse SVS I-III are the substrates for transglutaminase 4 (TGM4), a cross-linking enzyme secreted from the anterior prostate. According to activity assays, mouse TGM4 prefers a mild reducing and alkaline environment. However, under these conditions, the activity of mouse TGM4 toward SVS I-III was much lower than that of a common tissue-type TGM, TGM2. On the other hand, mouse TGM4 exhibited much higher cross-linking activity than TGM2 when native HMWCs containing SVS I-III were used as substrates under non-reducing condition. By the action of TGM4, the clot of SVS became more resistant to proteolysis. This indicates that the activity of TGM4 can further rigidify the copulatory plug and extend its presence in the female reproductive tract. Together with the properties of TGM4 and the nature of its disulfide-linked SVS protein substrates, male mice can easily transform the semen into a rigid and durable copulatory plug, which is an important advantage in sperm competition.     

Conserved sequences flank variable tandem repeats in two alleles of the G surface protein of Paramecium primaurelia

We describe the cloning and the sequencing of a macronuclear DNA fragment of Paramecium primaurelia, strain 168, encompassing the entire coding region of the 168G surface protein gene. Comparison of its nucleotide and its deduced amino acid sequences to those of the allelic surface protein 156G, previously described, reveals the rigorous conservation of a highly periodic structure. This structure is based on the presence of 37 periods of about 75 residues, each period containing eight cysteine residues. The differences between the two proteins are clustered in the central part of the sequence, which is itself made of quasi-identical tandem repeats. We propose that these repeats constitute the domain exposed on the surface of the cells and present the characteristics of concerted evolution.     

Evidence for mouse sulfhydryl oxidase‐assisted cross‐linking of major seminal vesicle proteins

Copulatory plug formation in animals is a general phenomenon by which competition is reduced among rival males. In mouse, the copulatory plug formation results from the coagulation of highly viscous seminal vesicle secretion (SVS) that is rich in proteins, such as dimers of SVS I, SVS I + II + III, and SVS II. These high‐molecular‐weight complexes (HMWCs) are also reported to be the bulk of proteins in the copulatory plug of the female mouse following copulation. In addition, mouse SVS contributes to the existence of sulfhydryl oxidase (Sox), which mediates the disulfide bond formation between cysteine residues. In this study, flavin adenine dinucleotide (FAD)‐dependent Sox was purified from mouse SVS using ion exchange and high‐performance liquid chromatography. The purified enzyme was identified to be Sox, based on western blot analysis with Sox antiserum and its capability of oxidizing dithiothreitol as substrate. The pH optima and thermal stability of the enzyme were determined. Among the metal ions tested, zinc showed an inhibitory effect on Sox activity. A prosthetic group of the enzyme was identified as FAD. The K_m and V_max of the enzyme was also determined. In addition to purification and biochemical characterization of seminal vesicle Sox, the major breakthrough of this study was proving its cross‐linking activity among SVS I–III monomers to form HMWCs in SVS.     

Molecular cloning and sequencing of the extracellular pectate lyase II gene from Erwinia carotovora Er.

Erwinia carotovora Er produces three extra-cellular pectate lyases (PL I, II, and III). The gene for pectate lyase II (pelII) of E. carotovora Er was cloned and expressed both in Escherichia coli and E. carotovora Er. Localization experiments in E. coli showed that PL II was exclusively in the cytoplasmic space, while PL II was excreted into the culture medium. The complete nucleotides of the pelII gene were sequenced and found to include one open reading frame of 1122 bp coding for a protein of 374 amino acid residues. From comparison of the N-terminal amino acid sequence between the purified PL II and the deduced protein from the nucleotide sequence we reached the conclusion that the mature protein is composed of 352 amino acids with a calculated molecular weight of 38,169 and is preceded by a typical signal sequence of 22 amino acid residues. PL II had 90.1% and 82.9% homologies with PL I and PL III in amino acid sequence, respectively.     

Analysis of the major large polypeptides of rat seminal vesicle secretion: SVS I, II, and III

Rat seminal vesicle secretion (SVS) contains a variety of protein complexes that seem to be linked by interchain disulfide bonds. Upon reduction and analysis by sodium dodecyl sulfate (SDS) gel electrophoresis, this pattern resolves to 3 major high molecular weight (SVS I-100,000, SVS II-50,000, SVS III-37,000) and 3 major low molecular weight protein bands (SVS IV, V, and VI). A two-dimensional SDS gel (1st dimension unreduced, 2nd dimension reduced) permitted identification of the components of the cross-linked species. In the native secretion, SVS I forms a series of oligomers that include both SVS II and III. Essentially all of SVS III is involved in these complexes, while the bulk of SVS II occurs instead as an apparent homodimer. The smaller proteins (SVS IV-VI) are not involved in covalently crosslinked complexes. The reduced forms of the larger polypeptides were isolated by a variety of procedures involving agarose gel filtration in 6M guanidine hydrochloride, reversed-phase high pressure liquid chromatography, ammonium sulfate fractionation, and preparative polyacrylamide gel electrophoresis. Based on its size, solubility, and amino acid composition, SVS II was identified as the major clottable protein of the secretion.     

Structure and evolution of the human involucrin gene

Involucrin is a keratinocyte protein that first appears in the cell cytosol, but ultimately becomes cross-linked to membrane proteins by transglutaminase. The gene for human involucrin has now been cloned and sequenced. The central segment of the coding region contains 39 repeats of a 30 nucleotide sequence whose ten encoded amino acids include three glutamines and two glutamic acids. This segment must have originated by successive duplications. Later duplications of modified sequences within the central segment can also be identified. Flanking the central segment lie shorter coding segments, a part of which must have given rise to the central segment. The flanking segments also show homology to a simpler 30 nucleotide sequence from which they likely originated. The evolution of involucrin as a substrate of transglutaminase and an envelope precursor was evidently made possible by this process of repeated mutation and duplication.     

Identification and characterization of tandem repeats in exon III of dopamine receptor D4 (DRD4) genes from different mammalian species

In this study we have identified and characterized dopamine receptor D4 (DRD4) exon III tandem repeats in 33 public available nucleotide sequences from different mammalian species. We found that the tandem repeat in canids could be described in a novel and simple way, namely, as a structure composed of 15- and 12- bp modules. Tandem repeats composed of 18-bp modules were found in sequences from the horse, zebra, onager, and donkey, Asiatic bear, polar bear, common raccoon, dolphin, harbor porpoise, and domestic cat. Several of these sequences have been analyzed previously without a tandem repeat being found. In the domestic cow and gray seal we identified tandem repeats composed of 36-bp modules, each consisting of two closely related 18-bp basic units. A tandem repeat consisting of 9-bp modules was identified in sequences from mink and ferret. In the European otter we detected an 18-bp tandem repeat, while a tandem repeat consisting of 27-bp modules was identified in a sequence from European badger. Both these tandem repeats were composed of 9-bp basic units, which were closely related with the 9-bp repeat modules identified in the mink and ferret. Tandem repeats could not be identified in sequences from rodents. All tandem repeats possessed a high GC content with a strong bias for C. On phylogenetic analysis of the tandem repeats evolutionary related species were clustered into the same groups. The degree of conservation of the tandem repeats varied significantly between species. The deduced amino acid sequences of most of the tandem repeats exhibited a high propensity for disorder. This was also the case with an amino acid sequence of the human DRD4 exon III tandem repeat, which was included in the study for comparative purposes. We identified proline-containing motifs for SH3 and WW domain binding proteins, potential phosphorylation sites, PDZ domain binding motifs, and FHA domain binding motifs in the amino acid sequences of the tandem repeats. The numbers of potential functional sites varied pronouncedly between species. Our observations provide a platform for future studies of the architecture and evolution of the DRD4 exon III tandem repeat, and they suggest that differences in the structure of this tandem repeat contribute to specialization and generation of diversity in receptor function.     

Molecular cloning and characterization of the circumsporozoite protein gene of Plasmodium inui isolated from Formosan macaques (Macaca cyclopis) in Taiwan

We characterized the complete nucleic and amino acid sequences of the Plasmodium inui circumsporozoite protein (Pincsp) gene and analyzed nucleotide diversity across the entire Pincsp gene by using 7 field isolates and strains Taiwan I and II obtained from Formosan macaques (Macaca cyclopis) in Taiwan. The length of the circumsporozoite protein ( CSP ) gene ranged from 1077 to 1125 bp. Size polymorphisms were due to variations in the number of tandem repeat units. The non-repetitive (NR) region exhibited high homology (99.1 ～ 100 and 98.7 ～ 100% at the nucleotide and amino acid levels, respectively) and was conserved among the variants (nucleotide diversities, π, of the 5'NR and 3'NR regions were 0.00364 and 0.00392, respectively). In the central repetitive (CR) region, we decomposed the sequences into 2 kinds of repeating amino acid motifs, i.e., a repeat unit R1, PA(P/A)(P/A)A(E)GG (n = 11-13), and a following repeat unit R2: P(A/G)(A/P/G)(P/Q)AQ(N/K) (n = 9-10). Analyzing these repeat sequences showed evidence of 3 genetic mechanisms for generating variations in the repeats of the Pincsp gene, i.e., point mutation, insertion, and recombination. These findings suggest that polymorphisms in the Pincsp gene are essentially limited to the CR region, which showed much greater variability in terms of length, number of repeats, and sequence.     

Characterization by cDNA cloning of the mRNA for seminalplasmin, the major basic protein of bull semen

A cDNA library derived from poly(A)+RNA of bull seminal vesicle tissue was screened with synthetic DNA probes specific for seminalplasmin (SAP), the major basic protein of bull semen. From a number of positive clones, pBSV12, containing a 577-bp insert, was identified and sequenced. The derived amino acid sequence comprises the known amino acid sequence of SAP with an amino terminal representing a putative signal sequence; at the carboxyl terminus the sequence contains an additional lysine residue. Present experimental data do not distinguish between two potential SAP precursor molecules, each starting with a methionine residue and differing by 10 amino acid residues in the leader peptide. Comparative Northern analysis reveals a SAP-specific mRNA of 700 bp, which lacks RNA from bovine testis as well as from seminal vesicle tissue of a bull calf; hence, expression of the SAP gene appears to be under androgen and/or developmental control. Southern analysis indicates that one gene appears to specify SAP. SAP-like DNA sequences were detected in ovine and porcine genomic DNA.     

Structural analysis of mouse S-antigen

Mouse S-antigen clones were isolated from a mouse retinal cDNA library using a bovine S-antigen cDNA probe. The largest clone (MSC-242) comprised 1532 bp and contained the entire coding sequence. The nucleotide sequence homology between the mouse and bovine coding regions was 84%, while non-coding regions appeared to be more divergent. The deduced amino acid sequence indicated that the mouse S-antigen had 403 residues and its molecular ratio was 44,930. An overall amino acid sequence similarity of 84% was observed between the mouse and bovine proteins. This degree of similarity dropped to 60% and 47% at the N and the C termini, respectively. The local homology with alpha-transducin observed in the bovine proteins, including the putative phosphoryl and rhodopsin binding sites, was conserved in the mouse as well. There was no overall sequence similarity with other proteins listed in the National Biomedical Research Foundation (NBRF) protein sequence database. Among the uveitopathogenic sites for experimental autoimmune uveitis (EAU), peptides N and M were identical to their bovine counterparts. Peptides 3 and K, however, were more divergent. The short repeats within these peptides were conserved.