Identification and characterization of the Atlantic salmon peptide transporter 1a

Abstract

Peptide transporter 1 (PepT1) mediates the uptake of dietary di-/tripeptides in vertebrates. However, in teleost fish gut, more than one PepT1-type transporter might operate, because of teleost-specific whole gen(om)e duplication event(s) that occurred during evolution. Here, we describe a novel teleost di-/tripeptide transporter, i.e., the Atlantic salmon ( Salmo salar) peptide transporter 1a [PepT1a; or solute carrier family 15 member 1a (Slc15a1a)], which is a paralog (77% similarity and 64% identity at the amino acid level) of the well-described Atlantic salmon peptide transporter 1b [PepT1b, alias PepT1; or solute carrier family 15 member 1b (Slc15a1b)]. Comparative analysis and evolutionary relationships of gene/protein sequences were conducted after ad hoc database mining. Tissue mRNA expression analysis was performed by quantitative real-time PCR, whereas transport function analysis was accomplished by heterologous expression in Xenopus laevis oocytes and two-electrode voltage-clamp measurements. Atlantic salmon pept1a is highly expressed in the proximal intestine (pyloric ceca ≈ anterior midgut > midgut >> posterior midgut), in the same gut regions as pept1b but notably ~5-fold less abundant. Like PepT1b, Atlantic salmon PepT1a is a low‐affinity/high‐capacity system. Functional analysis showed electrogenic, Na+-independent/pH-dependent transport and apparent substrate affinity ( K0.5) values for Gly-Gln of 1.593 mmol/L at pH 7.6 and 0.076 mmol/L at pH 6.5. In summary, we show that a piscine PepT1a-type transporter is functional. Defining the role of Atlantic salmon PepT1a in the gut will help to understand the evolutionary and functional relationships among peptide transporters. Its functional characterization will contribute to elucidate the relevance of peptide transporters in Atlantic salmon nutritional physiology.

Effects of Short-Term Fasting on mRNA Expression of Ghrelin and the Peptide Transporters PepT1 and 2 in Atlantic Salmon (Salmo salar)

“…Pept1 is an important intestinal transporter of di- and tripeptides characterized by an expression largely varying during ontogeny, in response to nutritional status (for, e.g., food limitation/deprivation and fasting/refeeding), dietary amino acids, environmental conditions, and under certain disease state (such as gut inflammation) ( Amberg et al, 2008 ; Bakke et al, 2010 ; Verri et al, 2010 , 2017 ; Bucking and Schulte, 2012 ; Romano et al, 2014 ). The rostro-caudal gene expression analysis along the GIT (high expression in PC, AMG, and MG) and the expression ratio levels of both paralogous genes ( slc15a1b mRNA expression levels largely exceeding those of slc15a1a ) are in line with previous studies on Atlantic salmon ( Rønnestad et al, 2010 ; Gomes et al, 2020 ) and other salmonids such as rainbow trout ( Ostaszewska et al, 2010 ; Kamalam et al, 2013 ). In our study, 4 days of fasting induced a moderate tendency of downregulation of both slc15a1 paralogous genes along the GIT, with statistical differences in the PC for slc15a1a and in PC and MG for slc15a1b .…”

“…The emerging slc15a1a paralog has just recently been identified, and therefore, limited information is still available for this gene. So far, only two studies on teleost species are found, i.e., in zebrafish ( Vacca et al, 2019 ) and in Atlantic salmon ( Gomes et al, 2020 ). Consequently, the largest part of the data available so far on teleost species about the functional role of Pept1 proteins refers to the Pept1b type ( Verri et al, 2010 ; Romano et al, 2014 ; Verri et al, 2017 ).…”

Functional characterization of Atlantic salmon (Salmo salar L.) PepT2 transporters

“…Atlantic salmon were reared (Cargill Innovation Center, Dirdal, Norway) following standard procedures, in seawater at 8.7 °C with a stock density below 25 kg fish/m 3 and constant day light. The fish were fed a commercial EWOS 10 mm pellet size diet [for information regarding diet composition please refer to (Gomes et al ., 2020)] using automatic feeders 3 times a day (22:00-24:00, 02:00-04:00, 06:00-08:00). Atlantic salmon (906 ± 122 g wet weight; 38.8 ± 1.8 cm total length; n = 8) were collected 2 hours after the 06:00-08:00 meal and euthanized with an overdose of MS222 (tricaine methansulfonate 300 mg/L; Norsk Medisinaldepot AS, Bergen, Norway) on site.…”

“…In this paper, we report the characterization of two newly identified PepT2-type proteins in the Atlantic salmon ( Salmo salar , L.). Both transporters structurally and functionally resemble the mammalian PepT2 paradigm, thus differing from the two PepT1-type proteins previously described in salmon (Ronnestad et al ., 2010; Gomes et al ., 2020). The kinetic properties of the two Atlantic salmon PepT2 transporters have been explored by expressing the proteins in Xenopus laevis oocytes and then analyzed by two-electrode voltage-clamp recordings of the transport current ( I t) and the presteady-state current ( I PSS ).…”

Amino Acid Carriers of the Solute Carrier Families 7 (SLC7) and 38 (SLC38) Are Involved in Leucine Sensing in the Brain of Atlantic Salmon (Salmo salar)

“…Comparative sequence analysis was performed as previously outlined (Rønnestad et al, 2010;Verri and Werner, 2019;Gomes et al, 2020). Briefly, the paralogues of LAT1-, SNAT2-, and Thymallus thymallus, Hucho taimen) and an esociform (Esox lucius) were identified by BLAST against several gen(om)es using the National Center for Biotechnology Information (NCBI) databases (for details see Supplementary Table 1).…”