Isolation,expression and characterization of carp retinol-binding protein

Vitamin A alcohol and its precursors carotenoids are introduced in the organism with the diet, transported to the liver and from there as retinol to target tissues by a specific carrier, the retinol-binding protein (RBP). RBP, isolated and characterized in many vertebrates, shows very high homology among the species investigated; however, very little is known in fish. In the present work RBP cDNA isolated from a carp liver library was transcribed and translated in vitro and the corresponding protein characterized. Carp RBP amino acid sequence and tertiary structure are highly conserved, but the protein shows two peculiar and unique characteristics: the signal sequence is not processed by the ER signal peptidase and two N-glycosylations are present at the N-terminus portion of the protein. It was also demonstrated that RBP glycosylation is not a feature common to all teleosts. Transfection experiments show that the green fluorescent protein (GFP) can be directed into the secretory pathway by the carp RBP N-terminal region, both in fish and in mammal cells, demonstrating that the sequence, although not processed, is recognized as a secretory signal in different species. Results obtained from different investigators indicated that in fish plasma RBP circulates without interacting with transthyretin (TTR) or other proteins, suggesting that the complex with TTR, whose postulated function is to hamper easy kidney filtration of circulating RBP, has evolved later in the evolutionary scale. This hypothesis is reinforced by the finding that carp RBP, as well as trout and other lower vertebrates in which circulating complex has never been demonstrated, lacks a short C-terminal sequence that seems to be involved in RBP-TTR interaction. In carp, carbohydrates could be involved in the control of protein filtration through the kidney glomeruli. Moreover, experiments of carp RBP expression in Cos-1 cells and in the yeast Saccharomyces cerevisiae show that glycosylation is necessary for protein secretion; in particular, additional in vitro experiments have shown it is involved in protein translocation through ER membranes.     

Glycosylation at specific sites of erythropoietin is essential for biosynthesis, secretion, and biological function

The glycoprotein hormone erythropoietin (Ep), the primary regulator of erythropoiesis, is synthesized by the kidney and secreted as the mature protein with three N-linked and one O-linked oligosaccharide chains. To investigate the role(s) of each carbohydrate moiety in the biosynthesis and function of Ep, we have used oligonucleotide-directed mutagenesis of a cDNA for human Ep to alter the amino acids at each of the carbohydrate attachment sites. Each mutated cDNA construct was expressed in stably transfected sublines of a kidney cell line, baby hamster kidney. We show, by preventing attachment of N-linked carbohydrate at asparagines 38 or 83, or preventing O-linked glycosylation at serine 126, that glycosylation of each of these specific sites is critical for proper biosynthesis and secretion of Ep. Fractionation of cellular extracts demonstrated that the mutant proteins lacking glycosylation at each of these three sites, (38, 83, and 126) were associated mainly with membrane components or were degraded rapidly. Less than 10% of these three mutant proteins were processed properly and secreted from the cells. The Ep protein lacking N-linked glycosylation at asparagine 24 is synthesized and secreted as efficiently as native Ep. The carbohydrates at positions 24 and 38 may be involved in the biological activity of Ep, since the absence of either of the oligosaccharide side chains at these positions reduced the hormone's biological activity.     

Characterization, primary structure, and evolution of lamprey plasma albumin.

The most abundant protein found in blood plasma from the sea lamprey (Petromyzon marinus) has the hallmarks of a plasma albumin: namely, high abundance, solubility in distilled water, a small number of tryptophans, and a high content of cysteines and charged residues. As in other vertebrate albumins, not all the cysteines are disulfide bonded. An unusual feature of this protein is its molecular weight of 175,000, roughly 2.5 times the size of other vertebrate albumins. Its amino acid sequence, deduced from a series of overlapping cDNA clones, can be aligned with other members of the gene family including plasma albumin, alpha-fetoprotein, and vitamin-D binding protein, confirming that it is indeed an oversized albumin. An unusual feature of the sequence is a 28-amino acid stretch consisting of a serine-threonine repeat with the general motif (STTT). Lamprey albumin contains a 23-amino acid putative signal peptide and a 6-residue putative propeptide, which, when cleaved, yield a mature protein of 1,394 amino acids with a calculated molecular weight of 157,000. The sequence also includes nine potential N-linked glycosylation sites (Asn-X-Ser/Thr), consistent with observation that lamprey albumin is a glycoprotein. If all the potential glycosylation sites were occupied by clusters of 2,000 molecular weight each, the total molecular weight would be 175,000. Like other members of the gene family, lamprey albumin is composed of a series of 190-amino acid repeats, there being seven such domains all together. Quantitative amino acid sequence comparisons of lamprey albumin with the other members of the gene family indicate that it diverged from an ancestral albumin prior to the gene duplications leading to this diverse group. This notion is confirmed by the pattern of amino acid insertions and deletions observed in a consideration of all domains that compose this family. Furthermore, it suggests that the invention of albumin antedates the vertebrate radiation.     

The primary structure of piscine (Oncorhynchus mykiss) retinol-binding protein and a comparison with the three-dimensional structure of mammalian retinol-binding protein.

1. The primary structures of two variants of rainbow trout (Oncorhynchus mykiss) plasma retinol-binding protein (RBP) were determined and found to be approximately 60% identical with those of both human and Xenopus laevis RBPs. The comparable sequence similarities that we have found agree with the estimate of similar divergence times between bony fishes and mammals and between bony fishes and amphibians. The two piscine RBP variants differ by six amino acid substitutions at positions that are not crucial for the interaction with retinol, on the basis of the human RBP three-dimensional structure [Cowan, S. W., Newcomer, M. E. & Jones, T. A. (1990) Proteins Struct. Func. Genet. 8, 44-61]. 2. Models were developed for the three-dimensional structures of rainbow trout and X. laevis RBPs, based on that of human RBP. The overall three-dimensional structure appears to be very well preserved for RBPs isolated from vertebrate species for which the divergence time is 350-400 million years. At variance with an almost absolute conservation for the residues that participate in the formation of the retinol binding site in mammalian RBPs, several amino acid replacements are found for this part of the RBP molecule when the comparison is extended to piscine and amphibian RBPs. However, the only allowed amino acid replacements are either conservative or more than 0.4 nm distant from retinol. Besides the retinol binding site, a few regions at the protein surface appear to be rather conserved during phylogenetic development of vertebrates and, therefore, might be involved in molecular interactions.     

Cloning of a Putative Neuronal Nicotinic Acetylcholine Receptor Subunit

A cDNA clone was isolated from a rat superior cervical ganglion cDNA library with an oligonucleotide that hybridized to muscle-like nicotinic acetylcholine receptor (nAChR) subunit cDNA. The deduced amino acid sequence possesses characteristics expected of a nAChR subunit that does not bind acetylcholine, in addition to distinctive features such as unique cysteine residues and N-linked glycosylation sites.     

Evaluation of human N-linked glycosylation sites in murine granulocyte-macrophage colony-stimulating factor.

Nonglycosylated murine and human granulocyte-macrophage colony-stimulating factor have a molecular mass of approximately 14.5 kDa predicted from the primary amino acid sequence. The expression of both proteins in COS cells leads to a heterogeneous population of molecules that differ in the degree of glycosylation. Both human and murine molecules contain two N-linked glycosylation sites that are situated in nonhomologous locations along the linear sequence. Despite this difference both proteins show a similar size distribution among the glycosylation variants. These studies analyze the effects of introducing in the murine protein novel N-linked glycosylation sites corresponding to those sites found in the human molecule. A panel of molecules composed of various combinations of human N-linked glycosylation sites in either the presence or the absence of murine N-linked glycosylation was compared. Substitution of a proper human N-linked glycosylation consensus sequence at Asn 24 did not result in N-linked glycosylation, nor was there any considerable effect on bioactivity. Replacement of the N-linked glycosylation consensus sequence at Asn 34 results in glycosylation similar to that found in the human molecule and causes a significant decrease in bioactivity. These data suggest that the position of N-linked glycosylation is critical for maximal bioactivity in a particular species and that the changes in position of these sites in different species probably occurred during evolution in response to changes in their receptors.     

Glycosylation is essential for translocation of carp retinol-binding protein across the endoplasmic reticulum membrane

Retinoid transport is well characterized in many vertebrates, while it is still largely unexplored in fish. To study the transport and utilization of vitamin A in these organisms, we have isolated from a carp liver cDNA library retinol-binding protein, its plasma carrier. The primary structure of carp retinol-binding protein is very conserved, but presents unique features compared to those of the correspondent proteins isolated and characterized so far in other species: it has an uncleavable signal peptide and two N-glycosylation sites in the NH(2)-terminal region of the protein that are glycosylated in vivo. In this paper, we have investigated the function of the carbohydrate chains, by constructing three mutants deprived of the first, the second or both carbohydrates. The results of transient transfection of wild type and mutant retinol-binding protein in Cos cells followed by Western blotting and immunofluorescence analysis have shown that the absence of both carbohydrate moieties blocks secretion, while the presence of one carbohydrate group leads to an inefficient secretion. Experiments of carp RBP mRNA in vitro translation in a reticulocyte cell-free system in the presence of microsomes have demonstrated that N-glycosylation is necessary for efficient translocation across the endoplasmic reticulum membranes. Moreover, when Cos cells were transiently transfected with wild type and mutant retinol-binding protein (aa 1-67)-green fluorescent protein fusion constructs and semi-permeabilized with streptolysin O, immunofluorescence analysis with anti-green fluorescent protein antibody revealed that the double mutant is exposed to the cytosol, thus confirming the importance of glycan moieties in the translocation process.     

Different roles of individual N-linked oligosaccharide chains in folding, assembly, and transport of the simian virus 5 hemagglutinin-neuraminidase.

The role of N-linked glycosylation in protein maturation and transport has been studied by using the simian virus 5 hemagglutinin-neuraminidase (HN) protein, a model class II integral membrane glycoprotein. The sites of N-linked glycosylation on HN were identified by eliminating each of the potential sites for N-linked glycosylation by oligonucleotide-directed mutagenesis on a cDNA clone. Expression of the mutant HN proteins in eucaryotic cells indicated that four sites are used in the HN glycoprotein for the addition of N-linked oligosaccharide chains. These functional glycosylation sites were systematically eliminated in various combinations from HN to form a panel of mutants in which the roles of individual carbohydrate chains and groups of carbohydrate chains could be analyzed. Alterations in the normal glycosylation pattern resulted in the impairment of HN protein folding and assembly which, in turn, affected the intracellular transport of HN. The severity of the consequences on HN maturation depended on both the number of deleted carbohydrate sites and their position in the HN molecule. Analysis of the reactivity pattern of HN conformation-specific monoclonal antibodies with the mutant HN proteins indicated that one specific carbohydrate chain plays a major role in promoting the correct folding of HN. Another carbohydrate chain, which is not essential for the initial folding of HN was found to play a role in preventing the aggregation of HN oligomers. The HN molecules which were misfolded, owing to their altered glycosylation pattern, were retained in the endoplasmic reticulum. Double-label immunofluorescence experiments indicate that misfolded HN and folded HN are segregated in the same cell. Misfolded HN forms disulfide-linked aggregates and is stably associated with the resident endoplasmic reticulum protein, GRP78-BiP, whereas wild-type HN forms a specific and transient complex with GRP78-BiP during its folding process.     

Effect of N-linked glycosylation on hepatic lipase activity

Hepatic lipase (HL) is a secretory protein synthesized in hepatocytes and bound to liver endothelium. Previous studies have suggested that HL N-linked glycans are required for catalytic activity. To directly test this hypothesis, Xenopus laevis oocytes were used to express native rat HL or HL lacking one or both N-linked glycosylation sites. The expressed and secreted native HL had an apparent molecular mass of 53 kDa, consistent with purified rat liver HL. The mutant lacking both glycosylation sites, while poorly secreted, had an apparent molecular mass of 48 kDa, the same size observed for HL after enzymatic removal of N-linked oligosaccharides. Mutants lacking one of the two sites were intermediate in size and showed reduced secretion. Each of these expressed and secreted proteins had full catalytic activity that was inhibited by antisera to rat HL. Thus, N-linked glycosylation of rat HL, while important to lipase secretion, is not essential for the expression of lipase activity.     

Functional role of N-linked glycosylation on the rat melanin-concentrating hormone receptor 1

Melanin-concentrating hormone (MCH) is known to act through two G-protein-coupled receptors MCHR1 and MCHR2. MCHR1 has three potential sites (Asn13, Asn16 and Asn23) for N-linked glycosylation in its extracellular amino-terminus which may modulate its reactivity. Site-directed mutagenesis of the rat MCHR1 cDNA at single or multiple combinations of the three potential glycosylation sites was used to examine the role of the putative carbohydrate chains on receptor activity. It was found that all three potential N-linked glycosylation sites in MCHR1 were glycosylated, and that N-linked glycosylation of Asn23 was necessary for full activity. Furthermore, disruption of all three glycosylation sites impaired proper expression at the cell surface and receptor activity. These data outline the importance of the N-linked glycosylation of the MCHR1.     

Molecular isolation and characterisation of carp transforming growth factor beta 1 from activated leucocytes

The transforming growth factor (TGF beta) family of proteins are a set of pleiotropic secreted signalling molecules with unique and potent immunoregulatory properties. In this study the molecular cloning of carp TGF beta 1 is reported. A partial cDNA of the TGF beta protein was initially identified from a cDNA pool, obtained by subtracting the cDNAs from Con A-induced carp head kidney leucocytes from uninduced carp head kidney leucocyte cDNA. The entire coding sequence was assembled by sequencing both ends of the cDNA clone by using an anchored PCR reaction. Sequence analysis revealed an ORF encoding a protein of 376 amino acids, containing the similar unique pattern of conserved cysteines (seven out of nine) in the cysteine knot structure which exists in all known TGF beta proteins. Compared with other animal TGF beta s, the cDNA clone shows approximately 59-42, 40-38 and 37-36% amino acid identity with TGF beta 1, TGF beta 3 and TGF beta 2 respectively. Carp TGF beta 1 is expressed at low levels in carp head kidney, spleen, egg and liver, whereas its messenger RNA level is increased after activation of the head kidney leucocytes with Con A. Sequence analysis and pattern of expression suggests that this is the carp TGF beta 1.     

The piscine plasma retinol-binding protein. Purification, partial amino acid sequence and interaction with mammalian transthyretin of rainbow trout (Oncorhynchus mykiss) retinol-binding protein.

1. Retinol-binding protein (RBP) has been isolated from the pooled plasma or rainbow trouts (Oncorhinchus mykiss) by gel filtration, hydrophobic interaction chromatography and ion-exchange chromatography. By this procedure two forms of the protein, both with a molecular mass (approximately 20 kDa) similar to that of mammalian RBP, were purified to homogeneity. Five amino acid substitutions have been found in the partial (about 60%) sequences of the two forms of trout RBP, which are presumably acetylated at their N terminus. The apparent participation of six conserved cysteines in the formation of disulphide bridges, as in human RBP, and the similarity (about 60%) of the amino acid sequence of trout and mammalian RBPs, indicate the existence of a similar overall structure organization in evolutionary distant RBPs. 2. Although the two forms of trout RBP are not physiologically involved in the formation of any protein--protein complex in plasma, they are capable of interacting with mammalian transthyretin, albeit with a binding affinity (K'd = 15-40 microM) considerably lower than that of mammalian RBP. Our data indicate that the two forms of trout RBP also possess the region that in mammalian RBP has the functional role of binding transthyretin. It is suggested that transthyretin (or a homologous protein) was modified, during phylogenetic development of the non mammalian vertebrates, to acquire a binding site for such a region of the RBP molecule.     

cDNA cloning and expression in Xenopus laevis oocytes of pig renal dipeptidase, a glycosyl-phosphatidylinositol-anchored ectoenzyme.

Clones expressing renal dipeptidase (EC 3.4.13.11) have been isolated from a pig kidney cortex cDNA library after employing the polymerase chain reaction technique to amplify a region of the dipeptidase cDNA. The complete primary sequence of the enzyme has been deduced from a full length cDNA clone. This predicts a protein of 409 amino acids, a cleavable N-terminal signal sequence of 16 residues and two N-linked glycosylation sites. At the C-terminus of the predicted sequence is a stretch of mainly hydrophobic amino acids which is presumed to direct the attachment of the glycosyl-phosphatidylinositol membrane anchor. Expression of the mRNA for pig renal dipeptidase in Xenopus laevis oocytes led to the production of a disulphide-linked dimeric protein of subunit Mr 48,600 which was recognized by a polyclonal antiserum raised to renal dipeptidase purified from pig kidney cortex. Bacterial phosphatidylinositol-specific phospholipase C released renal dipeptidase from the surface of the oocytes and converted the amphipathic detergent-solubilized form of the dipeptidase to a hydrophilic form, indicating that Xenopus laevis oocytes can process expressed proteins to their glycosyl-phosphatidylinositol anchored form.     

Molecular cloning, characterization and expression analysis of QM gene from grass carp (Ctenopharyngodon idellus) homologous to Wilms' tumor suppressor

QM, a novel gene that was originally identified as a tumor suppressor, has been cloned from species encompassing members of higher vertebrate, plant and fungal kingdoms, but it is not well documented in fish. In present study, a gene homologous to QM was obtained from grass carp (Ctenopharyngodon idellus) head kidney and spleen cDNA library. The full-length grass carp QM (GcQM) cDNA of 759 bp contains a short 5' UTR of 22 bp, a 3' UTR of 89 bp and an open reading frame of 648 nucleotides that translates into a 215-amino acid peptide with a molecular weight of 24.5 kDa. The predicted GcQM contains a series of functional motifs that belong to the QM family signature conserved among different species. Multiple alignment analysis reveals that GcQM shares an overall identity of 62.4% approximately 97.7% with other members of QM family. The fish QM has a closest genetic relationship to chicken homologue Jif-1. The GcQM expresses constitutively in spleen, heart and brain, and significantly up-regulated by Aeromonas hydrophila and grass carp haemorrhagic virus (GCHV) in head kidney, spleen and liver. The results suggest that grass carp QM homolog is an inflammatory stress inducible gene associated with anti-bacterial and viral defense, and it plays an important role in immune defense.     

Identification of the N-linked glycosylation sites of vitamin K-dependent carboxylase and effect of glycosylation on carboxylase function

The vitamin K-dependent carboxylase is an integral membrane protein which is required for the post-translational modification of a variety of vitamin K-dependent proteins. Previous studies have suggested carboxylase is a glycoprotein with N-linked glycosylation sites. In this study, we identify the N-glycosylation sites of carboxylase by mass spectrometric peptide mapping analyses combined with site-directed mutagenesis. Our mass spectrometric results show that the N-linked glycosylation in carboxylase occurs at positions N459, N550, N605, and N627. Eliminating these glycosylation sites by changing asparagine to glutamine caused the mutant carboxylase to migrate faster on SDS-PAGE gels, adding further evidence that these sites are glycosylated. In addition, the mutation studies identified N525, a site that cannot be recovered by mass spectroscopy analysis, as a glycosylation site. Furthermore, the potential glycosylation site at N570 is glycosylated only if all five natural glycosylation sites are simultaneously mutated. Removal of the oligosaccharides by glycosidase from wild-type carboxylase or by elimination of the functional glycosylation sites by site-directed mutagenesis did not affect either the carboxylation or epoxidation activity when the small FLEEL pentapeptide was used as a substrate, suggesting that N-linked glycosylation is not required for the enzymatic function of carboxylase. In contrast, when site N570 and the five natural glycosylation sites were mutated simultaneously, the resulting carboxylase protein was degraded. Our results suggest that N-linked glycosylation is not essential for carboxylase enzymatic activity but is important for protein folding and stability.