A naturally occurring deaminated neuraminic acid, 3-deoxy-D-glycero-D-galacto-nonulosonic acid (KDN). Its unique occurrence at the nonreducing ends of oligosialyl chains in polysialoglycoprotein of rainbow trout eggs

An unknown deaminated sialic acid has been isolated from Salmo gairdneri (rainbow trout) egg polysialoglycoprotein. A combination of structural methods including gas-liquid chromatography, chemical and enzymatic analyses, mass spectrometry, and 400-MHz 1H NMR spectroscopy was used to determine the structure as 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (= 3-deoxy-D-glycero-D-galacto-nonulosonic acid; KDN). This structure has been confirmed by comparison with a chemically synthesized authentic sample of KDN. The natural occurrence of deaminated sialic acid in biological material has not been previously reported. A series of KDN-containing oligosialic acids were isolated from the polysialoglycoprotein after pH 4.7-catalyzed hydrolysis. Structural studies including methylation analysis, mass spectrometry, 1H NMR spectroscopy, and chemical reactivity were also used to confirm the structures of the sialyloligosaccharides as KDN alpha 2[8NeuGc alpha 2-]n (n = 1-7). The exclusive location of KDN at the nonreducing termini in polysialoglycoproteins protects oligo(poly)sialyl chains from exosialidases. Terminal capping of these chains may be important in egg activation in salmonid fishes.     

Synthesis of neoglycoconjugates containing deaminated neuraminic acid (KDN) using rat liver alpha2,6-sialyltransferase

2-Keto-3-deoxy-D- glycero -D- galacto -nononic acid (KDN) was introduced into asialotransferrin and N -acetyllactosamine (LacNAc) from CMP-KDN by using rat liver Galbeta1-->4GlcNAc alpha2, 6- sialyltransferase to form KDN-transferrin and KDN-LacNAc. These structures contain terminal KDNalpha2-->6Gal-residues, a glycotope that has not yet been described in natural glycoconjugates. KDN was transferred to all four Gal residues in asialotransferrin by this enzyme. The incorporation efficiency of KDN from CMP-KDN into asialotransferrin was about half that of Neu5Ac from CMP-Neu5Ac, based on the V max/ K m values for these donor substrates, 0.0527 min-1and 0.119 min-1, respectively. The KDNalpha2-->6Gal linkage was resistant to exosialidase treatment, in contrast to the sensitivity of the Neu5Acalpha2-->6Gal linkage. Interestingly, Sambucus sieboldiana agglutinin (SSA) was shown to prefer KDN-transferrin to the corresponding Neu5Ac-transferrin, as estimated by slot-blot analysis. The use of an alpha2,6-sialyltransferase to synthesize neoglycoproteins containing KDN has not been previously reported. Their facile synthesis using CMP-KDN and sialyltransferases with different specificities offers new possibilities to study the function of neo-KDN- glycoconjugates, and to explore their use in glycotechnology.      

Isolation and characterization of deaminated neuraminic acid-rich glycoprotein (KDN-gp-OF) in the ovarian fluid of rainbow trout (Salmo gairdneri)

A novel highly acidic glycoprotein (deaminated neuraminic acid-rich glycoprotein; KDN-gp) was first discovered as an integral component of the vitelline envelope of rainbow trout eggs [Inoue, S., et al. (1988) Biochem. Biophys. Res. Commun. 153, 172-176]. Another member of this class of glycoprotein has now been found in the ovarian (or coelomic) fluid of ovulating rainbow trout. This ovarian fluid KDN-glycoprotein is designated as KDN-gp-OF and its amino acid and carbohydrate compositions were compared with those of the vitelline envelope KDN-gp (KDN-gp-VE). KDN-gp-OF was similar to KDN-gp-VE in the carbohydrate composition and molecular weight. However, a small but definite difference in amino acid composition and the molecular weight range was found between KDN-gp-OF and KDN-gp-VE. The results suggest that in KDN-gp-OF some peptide sequences presumably present at either the C- or N-terminus are deleted from KDN-gp-VE. Possible biological function of KDN-gp-OF is discussed.     

Deaminated neuraminic acid-rich glycoprotein of rainbow trout egg vitelline envelope. Occurrence of a novel alpha-2,8-linked oligo(deaminated neuraminic acid) structure in O-linked glycan chains

Deaminated neuraminic acid-rich glycoprotein (KDN-gp), first found and isolated from the vitelline envelope of rainbow trout eggs (Inoue, S., Kanamori, A., Kitajima, K., and Inoue, Y. (1988) Biochem. Biophys. Res. Commun. 153, 172-176), has been found to contain a number of O-linked glycan. Oligosaccharides were released by alkaline borohydride treatment of KDN-gp. Following fractionation by DEAE-Sephadex A-25 and thin-layer chromatography, a series of acidic oligosaccharides were obtained and analyzed for their chemical structures. The structure is based on composition analysis, methylation analysis, alkali-catalyzed "peeling" reactions, periodate oxidation, 400-MHz one- and two-dimensional 1H NMR spectroscopy, and molecular secondary ion mass spectrometry. The O-linked oligosaccharides isolated from KDN-gp have been shown to contain a common core trisaccharide Gal beta 1-3GalNAc alpha 1-3GalNAc in which the terminal Gal residue is blocked by a single residue of deaminated neuraminic acid (KDN) and the proximal GalNAc residue is substituted by alpha-2,8-linked oligo(KDN) chains. Structures of KDN-oligosaccharide chains in the glycoprotein are novel and expressed by the following general formula, where n = 0-5, for which data are available. [formula: see text]     

Opsonizing properties of natural antibodies of rainbow trout, Oncorhynchus mykiss (Walbaum)

In order to confirm previous observations in which a protective effect of rainbow trout natural antibodies against furunculosis was suspected, phagocytosis studies wereconducted in vitro , using combinations of rainbow trout sera with high or low levels of natural antibodies and active or inactivated complement as opsonizing factors. Opsonization was observed in all the cases where complement was present, and to a lesser degree with sera containing only natural antibodies. The results confirm the prime importance of the complement system and provide additional evidence for a possible role of natural antibodies in antimicrobial defences.     

Preparation of deacetyl-, lyso-, and deacetyl-lyso-GM(3) by selective alkaline hydrolysis of GM3 ganglioside

Three methods (using GM3 quantities ranging from a few milligrams to grams) have been developed to prepare, in high yield, the three derivatives of ganglioside GM3 [alpha-Neu5Ac-(2-3)-beta-Gal-(1-4)-beta-Glc-(1-1)-ceramide]: deacetyl-GM3 [alpha-Neu-(2-3)-beta-Gal-(1-4)-beta-Glc-(1-1)-ceramide], lyso-GM3 [alpha-Neu5Ac-(2-3)-beta-Gal-(1-4)-beta-Glc-(1-1)-sphingosine], and deacetyl-lyso-GM3 [alpha-Neu-(2-3)-beta-Gal-(1-4)-beta-Glc-(1-1)-sphingosine]. This is the first report of the preparation of lyso-GM3 by a one-pot reaction. We can now define the optimal conditions for the different preparations. Preparation of deacetyl-GM3: alkaline reagent, 2 M KOH in water; GM3 concentration, 33 mg/ml; reaction temperature, 90 degrees C; reaction time, 3.5 h; nitrogen atmosphere. Preparation of deacetyl-lyso-GM3: alkaline reagent, 8 M KOH in water; GM3 concentration, 10 mg/ml; reaction temperature, 90 degrees C; reaction time, 18 h; nitrogen atmosphere. Preparation of lyso-GM(3): alkaline reagent, 1 M sodium tert-butoxide in methanol; GM3 concentration, 10 mg/ml; reaction temperature, 80 degrees C; reaction time, 18 h; anhydrous conditions. The percentage yield of deacetyl-GM3 was 70;-75%, that of deacetyl-lyso-GM3 100%, and of lyso-GM3 36;-40%.Deacetyl-GM3, deacetyl-lyso-GM3, and lyso-GM3 were purified by column chromatography, and chemical structures were confirmed by electron spray-mass spectrometry.     

Isolation and preliminary characteristics of β-N-acetylglucosaminidase in the sperm of Siberian sturgeon (Acipenser baerii) and rainbow trout (Oncorhynchus mykiss)

The aim of this study was to characterize the enzyme β- N -acetyglucosaminidase (β-NAGase) in the milt and spermatozoa extracts from Siberian sturgeon and rainbow trout. After ion exchange chromatography one protein peak showed β-NAGase activity in sturgeon milt plasma and sperm extracts of both species. Surprisingly, two protein peaks showing β-NAGase activity were found in rainbow trout milt plasma. The molecular mass of β-NAGase was estimated by gel filtration as 127 kDa for rainbow trout spermatozoa, 271 kDa for sturgeon spermatozoa, and 74 kDa for milt plasma from both species. The kinetic parameters were determined for milt plasma and sperm extracts. The optimum pH of the β-NAGases was 3.8 for sturgeon milt plasma, 4.4 for sturgeon sperm extract, and 4.4–4.8 for milt plasma and sperm extract from rainbow trout. K _m value of the β-NAGases was 0.212, 0.563, 0.779 m m for sturgeon milt plasma, sturgeon sperm extract or rainbow trout extract, respectively. The β-NAGase from sperm extracts in both species showed 100% activity even after incubation at 56°C by 20 min, whereas its activity was decreased to 23% in sturgeon milt plasma and to 2% in trout milt plasma.     

Structure elucidation of neutral, di-, tri-, and tetraglycosylceramides from High Five cells: identification of a novel (non-arthro-series) glycosphingolipid pathway

The major neutral glycosphingolipids (GSLs) of High Five insect cells have been extracted, purified, and characterized. It was anticipated that GSLs from High Five cells would follow the arthro-series pathway, known to be expressed by both insects and nematodes at least through the common tetraglycosylceramide (Glcbeta1Cer --> Manbeta4Glcbeta1Cer [MacCer] --> GlcNAcbeta3Manbeta4Glcbeta1Cer [At(3)Cer] --> GalNAcbeta4- GlcNAcbeta3Manbeta4Glcbeta1Cer [At(4)Cer]). Surprisingly, the structures of the major neutral High Five GSLs already diverge from the arthro-series pathway at the level of the triglycosylceramide. Studies by one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy and electrospray ionization mass spectrometry (ESI-MS) showed the structure of the predominant High Five triglycosylceramide to be Galbeta3Manbeta4Glcbeta1Cer, whereas the predominant tetraglycosylceramide was characterized as GalNAcalpha4Galbeta3Manbeta4- Glcbeta1Cer. Both of these structures are novel products for any cell or organism so far studied. The GalNAcalpha4 and Galbeta3 units are found in insect GSLs, but always as the fifth and sixth residues linked to GalNAcbeta4 in the arthro-series penta- and hexaglycosylceramide structures (At(5)Cer and At(6)Cer, respectively). The structure of the High Five tetraglycosylceramide thus requires a reversal of the usual order of action of the glycosyltransferases adding the GalNAcalpha4 and Galbeta3 residues in dipteran GSL biosynthesis and implies the existence of an insect Galbeta3-T capable of using Manbeta4Glcbeta1Cer as a substrate with high efficiency. The results demonstrate the potential appearance of unexpected glycoconjugate biosynthetic products even in widely used but unexamined systems, as well as a potential for core switching based on MacCer, as observed in mammalian cells based on the common LacCer intermediate.     

Isolation, characterization, and distribution of two cDNAs encoding for growth hormone receptor in rainbow trout (Oncorhynchus mykiss)

Growth hormone (GH) plays important roles in a vast array of physiological processes, including growth, metabolism, and reproduction. In this study, cDNAs for two unique growth hormone receptor variants were cloned and sequenced from rainbow trout. The two cDNAs, one consisting of 2920 bp and the other of 2820 bp, share 87.2% identity in nucleotide sequence and 85.5% identity in deduced amino acid sequence and presumably arose through gene duplication. The cDNAs encode for putative 593- and 594-amino acid growth hormone receptors (designated GHR1 and GHR2, respectively), each containing a single transmembrane domain and other motifs characteristic of the receptor family. Both GHR1 and GHR2 mRNAs were present in all tissues examined. Trout GHR mRNAs are differentially expressed, both in terms of abundance among tissues and in terms of abundance within selected tissues. GHR1 was more abundant than GHR2 in the brain, whereas GHR2 was more abundant than GHR1 in pancreas and spleen. These findings expand our understanding of the evolution of the GH receptor family and suggest that independent mechanisms serve to regulate the tissue-specific expression of GHR mRNAs.     

Isolation and partial characterization of a non-thionein, zinc-binding protein from the liver of rainbow trout (Salmo gairdneri)

A Zn-binding protein (ZBP) was induced in the liver of Donaldson strain rainbow trout by 7 mg/kg i.p. injections of divalent Zn ions. ZBP synthesis was inhibited (74%) by cycloheximide. ZBP had a minimum molecular weight of 16,900 and was composed of two subunits with apparent molecular weights of 9650 and 6050. Purified ZBP contained 4% phenylalanine, 4% isoleucine, 5-6% leucine and 3.48% Zn. Each protein molecule bound nine Zn atoms and contained at least four sulfhydryl groups. Molecular weight, two subunits and the presence of aromatic amino acids suggested that ZBP was not metallothionein.     

Isolation and structural elucidation of a water-soluble polysaccharide (PS-I) of a wild edible mushroom, Termitomyces striatus

A heteropolysaccharide (PS-I), isolated from the hot aqueous extract of an edible mushroom, Termitomyces striatus, is composed of d-glucose, d-galactose, d-mannose and l-fucose in a molar ratio 2:1:1:1. Structural investigation of the native as well as the Smith-degraded polysaccharide was carried out using methylation analysis, periodate oxidation studies and 1D and 2D NMR spectroscopy, and the repeating unit of the polysaccharide is established as follows: [carbohydrate structure: see text]     

Complete primary structure of rainbow trout type I collagen consisting of alpha1(I)alpha2(I)alpha3(I) heterotrimers.

The subunit compositions of skin and muscle type I collagens from rainbow trout were found to be alpha1(I)alpha2(I)alpha3(I) and [alpha1(I)](2)alpha2(I), respectively. The occurrence of alpha3(I) has been observed only for bonyfish. The skin collagen exhibited more susceptibility to both heat denaturation and MMP-13 digestion than the muscle counterpart; the former had a lower denaturation temperature by about 0.5 degrees C than the latter. The lower stability of skin collagen, however, is not due to the low levels of imino acids because the contents of Pro and Hyp were almost constant in both collagens. On the other hand, some cDNAs coding for the N-terminal and/or a part of triple-helical domains of proalpha(I) chains were cloned from the cDNA library of rainbow trout fibroblasts. These cDNAs together with the previously cloned collagen cDNAs gave information about the complete primary structure of type I procollagen. The main triple-helical domain of each proalpha(I) chain had 338 uninterrupted Gly-X-Y triplets consisting of 1014 amino acids and was unique in its high content of Gly-Gly doublets. In particular, the bonyfish-specific alpha(I) chain, proalpha3(I) was characterized by the small number of Gly-Pro-Pro triplets, 19, and the large number of Gly-Gly doublets, 38, in the triple-helical domain, compared to 23 and 22, respectively, for proalpha1(I). The small number of Gly-Pro-Pro and the large number of Gly-Gly in proalpha3(I) was assumed to partially loosen the triple-helical structure of skin collagen, leading to the lower stability of skin collagen mentioned above. Finally, phylogenetic analyses revealed that proalpha3(I) had diverged from proalpha1(I). This study is the first report of the complete primary structure of fish type I procollagen.     

Seasonal occurrence of the infectious stage of proliferative kidney disease (PKD) and resistance of rainbow trout, Salmo gairdneri Richardson, to reinfection

The seasonality of proliferative kidney disease (PKD) in rainbow trout, Salmo gairdneri Richardson, at the American River Hatchery, California was found to be primarily dependent on the presence or absence of the infectious stage in the water supply. This was determined by introducing sentinel trout into the hatchery water supply on a monthly basis, followed by their transfer to the laboratory for subsequent holding in 18° C, pathogen-free water for one month prior to examination. These exposures demonstrated that infections were obtained from April through October at ambient temperatures 12–20° C. Trout which had recovered from clinical infections were found to be resistant to reinfection. Resistance was induced by active infection and not just previous exposure to the infectious stage. Trout surviving PKD were also found to harbour later sporogonic stages of the parasite for at least one year following initial infection, but fully formed spores, as judged by well-developed valves, were not observed.     

Effects of glutamine on growth performance,nutrient content,fatty acid profile,and blood parameters of rainbow trout (Oncorhynchus mykiss)

In this study, different amounts of glutamine were added to the diet of rainbow trout, and they were then fed for a period of 90 days. The current research investigated the effects of glutamine on various aspects of rainbow trout, including growth performance, condition factor, viscerosomatic index, hepatosomatic index, carcass composition, fatty acid profile, hematological parameters, and biochemical parameters. The study's findings revealed that adding glutamine to the diet of rainbow trout had a beneficial impact on their growth features. The rainbow trout group that was fed a 2% concentration of glutamine experienced the most notable increase in growth rate. A statistically significant difference in growth was observed among all groups (p < 0.05). Adding glutamine to the diet increased the amount of protein and decreased the fat content in the flesh of the fish. Glutamine exerted an influence on the blood and biochemistry parameters of fish, as well as their fatty acid composition. In conclusion, the inclusion of glutamine in the diet of fish could potentially enhance their immune system, improve the quality of their muscles, and enhance their growth performance.     

Identification, mapping, and genomic structural analysis of an immunoreceptor tyrosine-based inhibition motif-bearing C-type lectin from homozygous clones of rainbow trout (Oncorhynchus mykiss)

Utilizing a spleen-derived cDNA library and rapid amplification of cDNA 5' ends, we cloned a novel type II C-type lectin from two homozygous clones of rainbow trout. The cDNA is 2535 bp in length, and contains a 1017-bp open reading frame. From this sequence, a protein containing 339 amino acids (aa) was deduced. Using PSI-BLAST to search the GenBank database, the deduced protein is a C-type lectin, belonging to the type II membrane receptors. The protein contains four domains: an 87-aa N-terminal cytoplasmic domain, a 21-aa transmembrane domain, an 82-aa neck domain, and a 149-aa C-terminal C-type lectin domain. Two immunoreceptor tyrosine-based inhibition motifs (ITIMs) were located in the N-terminal cytoplasmic domain. RT-PCR results indicated that this gene is transcribed mainly in peripheral blood lymphocytes, spleen, kidney, and gill, and its expression in liver and intestine is weak. Monoclonal antibody 1.14 was used to isolate B cells from peripheral blood lymphocytes. Analysis revealed that this gene is highly expressed in B cells. Genomic DNA was amplified with long-template PCR and sequenced. The gDNA is 12.0 kb in length and contains nine exons and eight introns. The first intron of the genes from the OSU and AR clones differed in length. Based on this difference, the genotype of 69 doubled-haploid offspring of OSU and AR were screened. Subsequently, this gene was mapped on the rainbow trout linkage map to group XXI. Results of a Southern blot indicated that the gene ( TCL-2) exists as a single copy in the rainbow trout genome. The genomic structure, the deduced protein structure, the tissue expression pattern, as well as the phylogenetic analysis of the carbohydrate recognition domain based on the deduced amino acid sequence indicate that TCL-2 resembles CD72; however, the carbohydrate recognition domain sequences of TCL-2 and CD72 are highly diverged.     

Development and characterization of novel tetra‐, tri‐, and dinucleotide microsatellite markers in rainbow trout (Oncorhynchus mykiss)

We discuss the development and characterization of 40 polymorphic rainbow trout (Oncorhynchus mykiss) microsatellite loci. We used enriched libraries to isolate 14 dinucleotide, seven trinucleodide, eight compound di/tetranucleotide, and 11 tetranucleotide loci. These markers will be useful for selective breeding via marker‐assisted selection, population genetics studies, parentage analysis, and have already been used for genome mapping.