Determination of Full-Length cDNA Nucleotide Sequence of Equine Carbonic Anhydrase VI and Its Expression in Various Tissues

Ochiai, Hideharu; Kanemaki, Nobuyuki; Kamoshida, Shino; Murakami, Masaru; Ichihara, Nobutsune; Asari, Masao; Nishita, Toshiho

doi:10.1292/jvms.71.1233

Cited by 6 publications

(8 citation statements)

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“…Canine CA-VI mRNA signals were strong in the major salivary glands and weaker in the minor salivary glands and esophagus, and were absent in the pancreas, liver, and almost all parts of the digestive tract, except the esophagus [17]. In the horse, CA-VI mRNA was detected in the digestive tract, salivary glands, testis, thyroid gland, and liver, but not in nerve tissue, skeletal muscle, spleen, or lymph node [18]. …”

Section: Discussionmentioning

confidence: 99%

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Isolation and sequencing of swine carbonic anhydrase VI, an enzyme expressed in the swine kidney

et al. 2014

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BackgroundCarbonic anhydrase VI (CA-VI) is produced by the salivary gland and is secreted into the saliva. Although CA-VI is found in the epithelial cells of distal straight tubule of swine kidneys, the exact function of CA-VI in the kidneys remains unclear.ResultsCA-VI was located in the epithelial cells of distal straight tubule of swine kidneys.A full-length cDNA clone of CA-VI was generated from the swine parotid gland by reverse transcription polymerase chain reaction, using degenerate primers designed based on conserved regions of the same locus in human and bovine tissues.The cDNA sequence was 1348 base pairs long and was predicted to encode a 317 amino acid polypeptide with a putative signal peptide of 17 amino acids. The deduced amino acid sequence of mature CA-VI was most similar (77.4%) to that of human CA-VI. CA-VI expression was confirmed in both normal and nephritic kidneys, as well as parotid. As the primers used in this study spanned two exons, the influence of genomic DNA was not detected. The expression of CA-VI was demonstrated in both normal and nephritic kidneys, and mRNA of CA-VI in the normal kidneys which was the normalised to an endogenous β–actin was 0.098 ± 0.047, while it was significantly lower in the diseased kidneys (0.012 ± 0.007). The level of CA-VI mRNA in normal kidneys was 19-fold lower than that of the parotid gland (1.887).ConclusionsThe localisation of CA-VI indicates that it may play a specialised role in the kidney.

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Section: Discussionmentioning

confidence: 99%

“…To date, human, bovine, mouse, canine, and equine CA-VI cDNAs have been cloned successfully [14-18]. CA-VI was previously reported to be present in the parotid gland, saliva, bile, and serum of pigs [19].…”

Section: Introductionmentioning

confidence: 99%

Isolation and sequencing of swine carbonic anhydrase VI, an enzyme expressed in the swine kidney

et al. 2014

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“…Western blot analysis of LAT: The anti-canine LAT1 serum was prepared as described previously [26]. The cell membrane of the tissues for Western blot analysis was prepared as reported by Denker et al [7].…”

Section: Real-time Rt-pcr Evaluation Of Lat1mentioning

confidence: 99%

Analysis of L-type amino acid transporter in canine hepatocellular carcinoma

OGIHARA¹,

NAYA²,

Sato³

et al. 2015

The Journal of Veterinary Medical Science

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ABSTRACT. Analysis of L-type amino acid transport expression of hepatocellular carcinoma cells (HCCs) of the dog was performed. The leucine transport activity of canine HCCs was 0.628 ± 0.018 nmol/mg protein/min. The inhibitor of LAT 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH) reduced 90% of the activity at 1 mM. The deduced amino acid sequences of canine LAT2, LAT3 and LAT4 were well conserved in mammalians, exhibiting 89, 88 and 77% homology, respectively. RT-PCR revealed distinct LAT1 expression compared with normal hepatocytes. Western blotting analysis confirmed the potent LAT1 expression in canine HCCs but not hepatocytes, and realtime RT-PCR analysis indicated that canine HCCs possessed 28 times higher LAT1 expression than hepatocytes. These results indicated that the leucine transport activity of canine HCCs was due to LAT1. KEY WORDS: canine, hepatoma, LAT, transporter doi: 10.1292/jvms.14-0392; J. Vet. Med. Sci. 77(5): 527-534, 2015 Many amino acid transport systems have been distinguished based on differences in their substrate-selectivity, ion-dependence, pH sensitivity, kinetics and regulatory properties by using membrane vesicle preparations or cultured cells [5,6]. Among them, system L is an amino acid transport system that mediates sodium-independent transport of neutral amino acids. It was first described in Ehrlich ascites tumor cells as a sodium-independent transport system for neutral amino acids that is specifically inhibited by a bicyclic amino acid, 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH) [28,29,37]. Later, 2 subtypes for system L with distinct characteristics in substrate affinity, selectivity, and transport properties were reported and named systems L1 and L2 [37]. To date, 4 isoforms of system L have been identified at the molecular level. The first cloned transporters with system L activity were LAT1 and LAT2, which are members of the SLC (solute carrier) 7 family of transporters [17,21,34,36]. They mediate sodium-independent amino acid exchange and recognize a wide range of large neutral amino acids as substrates [35], expanding to small neutral amino acids in the case of LAT2 [30,31,33]. These proteins form heteromeric complexes via a disulfide bond with the heavy chain of 4F2 antigen (4F2hc, SLC3A2), a single transmembrane domain protein essential for the functional expression of LAT1 and LAT2.Later, other transporters were identified by expression cloning [1,3], and it was reported that they exhibited Naindependent, BCH-sensitive neutral amino acid transport activity. In spite of possessing the properties of system L, these transporters (LAT3 and LAT4) were structurally different from system L1 (LAT1 and LAT2), and additionally, they did not require co-expression with 4F2hc to elicit transport activity at the plasma membrane. LAT3 and LAT4 transport activities show specific properties: (i) preferential substrate specificity [6], (ii) complex kinetics [6], which are compatible with 2 simultaneous apparent affinities and (iii) inactivation by the thiol reagen...

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“…The 1 ml supernatant was laid over a 5 ml sucrose solution containing 0.8 mM sucrose and 2 mM Na-EGTA, and was centrifuged at 32,000 g for 40 min. Protein concentration of the pellet was determined by the BCA method, and was used for Western blot analysis as previously described [15]. The membrane-rich fraction was simply solubilized, electrophoresed into 12% polyacrylamide gels, and immunoblotted with a chemiluminescence autoradiograph.…”

Section: Western Blot Analysismentioning

confidence: 99%

Distribution, Molecular Structure and Functional Analysis of Carnitine Transporter (SLC22A5) in Canine Lens Epithelial Cells

et al. 2014

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While carnitine has been reported to have an anti-oxidative role on the ocular surface, there has been no report on the existence of a carnitine transporter (SLC22A5) in the lens. Therefore, we investigated the carnitine transport activity of canine lens epithelial cells (LEC) and determined the molecular structure of canine SLC22A5. The carnitine transport activity was 7.16 ± 0.48 pmol/ mg protein/30 min. Butyrobetaine, the analogue of carnitine, reduced 30% of the activity at 50 µM. A coding sequence of canine carnitine transporter was 1694 bp long and was predicted to encode 557 amino acid polypeptides. The deduced amino acid sequence of canine carnitine transporter showed >80% similarity to that of mouse and human. Western blot analysis detected the band at 60 kDa in the membrane of lens epithelial cells. The high content of carnitine in the lens is possibly transported from aqueous humor by SLC22A5.

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Determination of Full-Length cDNA Nucleotide Sequence of Equine Carbonic Anhydrase VI and Its Expression in Various Tissues

Cited by 6 publications

References 24 publications

Isolation and sequencing of swine carbonic anhydrase VI, an enzyme expressed in the swine kidney

Isolation and sequencing of swine carbonic anhydrase VI, an enzyme expressed in the swine kidney

Analysis of L-type amino acid transporter in canine hepatocellular carcinoma

Distribution, Molecular Structure and Functional Analysis of Carnitine Transporter (SLC22A5) in Canine Lens Epithelial Cells

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