cold adaptation strategy via a unique seven-amino acid domain in the icefish (Chionodraco hamatus) PEPT1 transporter

Antonia Rizzelloa,1, Alessandro Romanoa,1, Gabor Kottrab, Raffaele Aciernoa, Carlo Storellia, Tiziano Verria, Hannelore Danielb, and Michele Maffiaa,2

aLaboratory of General Physiology, Department of Biological and Environmental Sciences and Technologies, University of Salento, I-73100 Lecce, Italy; and bMolecular Nutrition Unit, Nutrition and Food Research Center, Technical University of Munich, D-85350 Freising-Weihenstephan, Germany

Edited by George N. Somero, Stanford University, Pacific Grove, CA, and approved February 13, 2013 (received for review November 27, 2012) Adaptation of organisms to extreme environments requires pro- PEPT1 [ 15 (oligopeptide transporter) teins to work at thermodynamically unfavorable conditions. To member 1; SLC15A1] is an integral of the brush adapt to subzero temperatures, increase the flexibility of border membrane of intestinal epithelial cells, which mediates the parts of, or even the whole, 3D structure to compensate for the uptake of di- and tripeptides derived from dietary protein hydro- lower thermal kinetic energy available at low temperatures. This lysis in the gut lumen. Since the identification of the first ortholog may be achieved through single-site amino acid substitutions in from the rabbit (21), numerous studies have addressed the mo- regions of the protein that undergo large movements during the lecular architecture as well as biochemical and physiological func- catalytic cycle, such as in enzymes or transporter proteins. Other tions of the proteins from a variety of vertebrate species, including strategies of cold adaptation involving changes in the primary fish (22–24). Thus, PEPT1 gives a molecular background to con- amino acid sequence have not been documented yet. In Antarctic duct comparative studies on the intrinsic functional and structural icefish (Chionodraco hamatus) peptide transporter 1 (PEPT1), the adaptation of a membrane transport protein to cold. first transporter cloned from a vertebrate living at subzero temper- To investigate the mechanisms of cold adaptation in a trans-

atures, we came upon a unique principle of cold adaptation. A de membrane protein, we cloned the transporter protein PEPT1 PHYSIOLOGY novo domain composed of one to six repeats of seven amino acids from the Antarctic icefish (a vertebrate living at subzero tem- (VDMSRKS), placed as an extra stretch in the cytosolic COOH-termi- peratures), and studied the structure–function relationship with nal region, contributed per se to cold adaptation. VDMSRKS was in respect to the temperature dependence of transport. We dis- a protein region uninvolved in transport activity and, notably, when covered that a unique domain of seven amino acids, placed as an transferred to the COOH terminus of a warm-adapted (rabbit) extra stretch in the COOH-terminal region of icefish PEPT1, PEPT1, it conferred cold adaptation to the receiving protein. Overall, contributes to the cold adaptation of this transporter. Trans- we provide a paradigm for protein cold adaptation that relies on fi ferring this domain into the COOH terminus of a warm-adapted insertion of a unique domain that confers greater af nity and max- transporter protein shifts its temperature dependence toward imal transport rates at low temperatures. Due to its ability to trans- that of the icefish protein. fer a thermal trait, the VDMSRKS domain represents a useful tool for future cell biology or biotechnological applications. Results Icefish PEPT1 COOH Terminus Comprises an Additional VDMSRKS Antarctic fish | SLC15A1 | membrane transport proteins | Domain, Which Is Repeated One to Six Times. The icefish pept1 temperature dependence cDNA was obtained using a PCR-based homology cloning strategy (Fig. S1). Icefish pept1 encoded a 757-amino acid protein, with 12 y living at subzero temperatures (−1.9 °C), Antarctic noto- transmembrane domains (TMDs) and a large extracellular loop Bthenioids provide an excellent opportunity to study the between TMDs IX and X (Fig. 1A). Icefish PEPT1 exhibited evolutionary and functional features of biochemical adaptation a higher percentage of identity with fish (66–68%) than avian to low temperatures (1, 2). Numerous studies have addressed the (62%) and mammalian (56–57%) PEPT1 transporters, and it question of how cytoplasmic enzymes evolved in these organisms clustered to the “fish” branch of the vertebrate PEPT1 phyloge- enabling proper cellular functions under these severe environ- netic tree (Fig. 1B). The tissue-specific expression of icefish pept1 mental conditions (1–6). mRNA resembled that previously reported for other vertebrate Membrane transport proteins also play a crucial role in main- PEPT1 transporters, showing the highest level of expression in taining cellular homeostasis, and, like enzymes, their catalytic ac- intestine and kidney (Fig. 1C). Icefish PEPT1 retained the typical tivity depends on temperature (7–13). However, how membrane features and the main structural/functional domains of the PEPT1- transport proteins properly control the influx and efflux of solutes type transporters [e.g., the PTR2 (peptide transporter 2) signature 1 across cellular membranes at low temperatures largely is unknown. In any event, both the intrinsic structural characteristics (i.e., amino acid sequence) and specific protein–lipid membrane interactions Author contributions: A. Rizzello, A. Romano, G.K., T.V., H.D., and M.M. designed re- may contribute, alternatively or in parallel, to the adaptive features search; A. Rizzello and A. Romano performed research; G.K., C.S., T.V., H.D., and M.M. contributed new reagents/analytic tools; A. Rizzello, A. Romano, G.K., and R.A. analyzed displayed by temperature-adapted transporters. Functional studies data; and A. Rizzello, A. Romano, G.K., R.A., C.S., T.V., H.D., and M.M. wrote the paper. + 2+ on cold-adapted transporters are limited to the Na /Ca exchanger The authors declare no conflict of interest. Oncorhynchus mykiss – of the rainbow trout ( )(14 16) and the sarco This article is a PNAS Direct Submission. 2+ (endo)plasmic reticulum Ca -ATPase (SERCA) of the cold-tol- Data deposition: The icefish PEPT1 cDNA sequence reported in this paper has been de- erant wood frog (Rana sylvatica) (17). Limited data also are avail- posited in the GenBank database (accession no. AY170828.2). able from the Antarctic emerald rockcod (Trematomus bernacchii) 1A. Rizzello and A. Romano contributed equally to this work. fi Chiono- sodium 1 (SGLT1) (18), the ice sh ( 2To whom correspondence should be addressed. E-mail: michele.maffi[email protected]. draco hamatus) peptide transporter 1 (PEPT1) (19), and, recently, + + This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the Antarctic octopus (Pareledone sp.) Na /K -ATPase (20). 1073/pnas.1220417110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1220417110 PNAS Early Edition | 1of6 Downloaded by guest on October 2, 2021 Fig. 1. Sequence and expression analysis of icefish PEPT1. (A) Model of icefish PEPT1. The picture was obtained using TeXtopo (47). TMDs I–XII were predicted using TMHMM (transmembrane prediction using hidden Markov models) 2.0. N-glycosylation (yellow ●), cAMP-dependent protein kinase A (PKA; purple ●), PKC (azure ●), and overlapped PKA/PKC (orange ●) sites; PTR2 family proton/oligopeptide symporters signature 1 (blue) and 2 (green) motifs were identified using PROSITE 20.24. The six VDMSRKS domains close to the COOH terminus are indicated alternately in red and pink. (B) Unrooted phylogenetic tree of vertebrate PEPT1 transporters. Alignments were performed using ClustalW2. The unrooted tree (1,000 times bootstrap) was constructed using neighbor-joining analysis with data corrected for multiple hits through Poisson distribution. (C) Tissue distribution of icefish pept1 mRNA. Expression levels of icefish pept1 mRNA in different tissues determined by qualitative RT-PCR (Upper) and real-time PCR (Lower) analysis. Relative values are expressed with respect to the intestinal signal. (D) Comparison of the COOH-terminal regions of PEPT1 from four Antarctic teleosts. The alignment (ClustalW2) indicates TMD XI and XII and the conserved seven-amino acid domains (D1–D6).

and 2 motifs and the TMDs] (Fig. S2). Interestingly, in the icefish found in icefish PEPT1, genomic DNA from several C. hamatus PEPT1 COOH-terminal region, six direct repeats of seven amino DNA samples were screened by PCR. The analysis revealed that (i) acids (VDMSRKS) were identified, perfectly conserved among the entire repeat motif was encoded within a single exon, homol- the repetitive units (Fig. 1A and Figs. S1 and S2). The VDMSRKS ogous to the last exon (exon 23) of other characterized vertebrate domain is not found in any other PEPT1 transporter, and no pept1 (Fig. S3)and(ii) this region is a polymorphic locus that significant similarities to any classified protein as deposited in presents several repeated domains, varying from one to six (Fig. S4 various databases were identified. Moreover, a search for poten- and Table S1). tial posttranslational modification sites identified in this domain a putative protein kinase C phosphorylation site (Fig. 1A and Fig. Icefish PEPT1 Protein Operates as a Canonical PEPT1-Type Transporter. S1). Analysis of the COOH-terminal region of PEPT1 trans- Icefish PEPT1 is an electrogenic symporter that transports various porters from different Antarctic teleosts (Histiodraco velifer, di- and tripeptides with distinct current–voltage relationships for Trematomus pennellii, and T. bernacchii) revealed that the seven- zwitterionic and charged peptides (Fig. 2A and Fig. S5). Apparent amino acid domain identified in icefish was conserved among the affinities for the model dipeptide glycyl-L-glutamine (GQ) are in the other cold-adapted species (Fig. 1D), but, unlike in icefish, in millimolar range (Fig. 2B). Kinetic parameters depend on mem- these species only a single unit of seven amino acids was observed. brane potential and extracellular pH (Fig. 2 C and D). Based on this To assess possible allelic variation in the number of repeats as basic phenotyping, we conclude that the icefish protein shows

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Fig. 2. Functional characterization of icefish PEPT1. (A) Steady-state current–voltage relationships for GQ. Icefish PEPT1-expressing oocytes were perfused at

pH 6.5 in the presence of increasing (0.05–5 mM) GQ concentrations. (B) Kinetic parameters of GQ transport. Apparent GQ affinity (K0.5) and maximal transport current (Imax) were calculated at −60 and −120 mV by least-squares fitting to the Michaelis-Menten equation. Imax values are expressed as the percentage of the value calculated at pH 6.5 in the same experiment. n, number of oocytes; N, number of frogs. (C and D) Membrane potential and pH

dependence of K0.5 and Imax of GQ transport. Imax values have been normalized to those measured at −160 mV.

a transport behavior very similar to that reported for other terminus (riPEPT1; Fig. 4A), rabbit PEPT1 acquires a clear vertebrate PEPT1 proteins (25–28); for a detailed discussion, cold-adapted phenotype with a Q10 of 4.32 ± 0.15 and an Ea of see SI Results. 106 ± 1kJ/mol(Fig.4B–D), and this thermal phenotype is abolished with the removal of the six heptad repeats from VDMSRKS Domain Contains Structural Determinants That Contribute the COOH terminus of the chimeric transporter (riPEPT1-Δ6; Fig. fi to Cold Adaptation of PEPT1-Type Transporters. Ice sh PEPT1 is 4A), leading to a Q10 of 5.18 ± 0.33 and an Ea of 127 ± 3kJ/mol functionally adapted to cold. In fact, the change in transport rate in (Fig. 4 B–D). This modification also changes only the thermal response to temperature, expressed by the Q10 value, is consider- parameters but not the transport kinetics (Table S2). ably lower (3.14 ± 0.33) than that of the warm-adapted rabbit (5.21 ± 0.58) or zebrafish (5.00 ± 0.33) PEPT1 proteins (Fig. 3 A Discussion and B). Moreover, the apparent activation energy (Ea), calculated Adaptation of integral membrane transport proteins to work at from the slope of the (invariably linear) Arrhenius plots, is mark- low temperatures is crucial to organismic homeostasis in cold- edly lower (79 ± 6 kJ/mol) than that of rabbit (133 ± 6 kJ/mol) and adapted vertebrates. Although a few studies have assessed mem- zebrafish (125 ± 6 kJ/mol) transporters (Fig. 3 C and D). Icefish brane transporter function in species living at low temperatures PEPT1’s adaptation to cold depends partly on its unique COOH- (14–19, 29), the underlying molecular structural features allowing terminal VDMSRKS domain. In fact, the icefish PEPT1 mutant these proteins to work at extreme temperatures remains unclear. lacking all six heptad repeats (icefish PEPT1-Δ6; Fig. 3A) displays With its 13-My–long confinement to cold waters, the endemic asignificant shift of its thermal parameters toward those of the Antarctic icefish C. hamatus is a strictly stenothermal vertebrate warm-adapted transporters (Q10 = 4.10 ± 0.20 and Ea = 102 ± 4kJ/ adapted to subzero temperatures (−1.9 °C). To investigate the mol; Fig. 3 B–D). The four icefish PEPT1 mutants corresponding to functional and structural features that confer cold adaptation to an the natural sequence variants observed in this study (icefish PEPT1- integral membrane transport protein in this psychrophilic model Δ5, -Δ4, -Δ3, and -Δ2; Fig. 3A), however, all retain the typical icefish vertebrate, we cloned the Antarctic icefish PEPT1-type trans- PEPT1 thermal response. All deletions carried out in the icefish porter, which is responsible for the intestinal uptake of di- and PEPT1 COOH terminus did not alter the transport kinetics (Fig. S6 tripeptides derived from dietary protein breakdown in an elec- + and Table S2), except for temperature dependence. Most inter- trogenic, H -coupled transport process. The choice of PEPT1 as estingly, the VDMSRKS repeat region also can modify the tem- a model transmembrane transport protein was based on the perature dependence of a warm-adapted transporter. When the availability of molecular, biochemical, and physiological data from COOH terminus of rabbit PEPT1 is replaced by the icefish COOH a variety of vertebrate species (for reviews see, e.g., refs. 22 and

Rizzello et al. PNAS Early Edition | 3of6 Downloaded by guest on October 2, 2021 Fig. 3. Temperature sensitivity of wild-type and mutant icefish PEPT1. (A) Schematic representation of wild-type and mutant PEPT1 transporters. (B) Q10 values of wild-type and mutant transporters. Q10 was calculated over the temperature range 280–300 K (7–27 °C) in the presence of saturating concentrations of GQ (20 mM). (C) Arrhenius plots of wild-type and mutant transporters. Transport currents were calculated at −60 mV and different temperatures (280–300

K). Currents were normalized to that at 300 K. Lines represent the linear regression of the transformed data. (D) Activation energy values (Ea) of wild-type and mutant transporters. Data were calculated from the slope of the curves reported in C. For all analyses, values were means ± SE (n = 6–9). a, difference vs. icefish PEPT1; b, difference vs. icefish PEPT1-Δ6 (one-way ANOVA/Bonferroni post hoc test). **P < 0.01.

23), allowing comparative studies on the intrinsic functional and enzymes (6, 30, 31) and has been found in other cold-adapted + + structural adaptation of this protein to cold. membrane transport proteins, such as the rainbow trout Na /Ca2 Overall, the analysis of the icefish PEPT1 established that it is exchanger (15) and the wood frog SERCA pump (17). It must be a “canonical” peptide transporter with all acknowledged features emphasized that our experimental setup with the use of Xenopus of vertebrate PEPT1-type transporters. Icefish pept1 mRNA is laevis oocytes as a heterologous expression system for all species highly expressed in intestine and kidney, as it is in mammals, and guarantees that icefish, rabbit, and zebrafish PEPT1 transporters the protein operates functionally as a classical PEPT1-type trans- are studied in the same lipid environment, so the observed dif- porter with respect to mode of transport, kinetics, membrane ferences in temperature dependence must originate from the in- potential dependence, pH dependence, electrogenicity, substrate trinsic properties of the proteins. affinity, and substrate specificity. The amino acid sequence of the Aspecific feature in the primary sequence of icefish PEPT1 is icefish PEPT1 protein possesses all primary structural prototypical a COOH-terminal polymorphic region consisting of one to six di- elements of a PEPT1-type transporter with respect to amino acids, rect-repeat domains of seven amino acids (VDMSRKS), perfectly motifs, and domains identified as relevant for function. However, conserved among the repetitive units. The entire polymorphic re- despite the high similarity to its warm-adapted orthologs, icefish gion is encoded within a single exon, homologous to the last exon PEPT1 shows a unique functional and structural adaptation phe- (exon 23) of the other PEPT1 genes characterized so far in verte- nomenon at low temperatures. The transporter is less temperature brates (32–35). This seven-amino acid domain is found invariably dependent than the orthologs from rabbit and zebrafish, and both conserved only in the COOH terminal region of PEPT1 proteins of Q10 and Ea values are significantly lower (Q10 = 3.14 and Ea = 79 other cold-adapted teleost fishes living in Antarctica (H. velifer, kJ/mol, respectively) than those measured for the warm-adapted T. pennellii and T. bernacchii). This sequence has not been de- proteins (Q10 = 5.21 and Ea = 133 kJ/mol for rabbit and Q10 = 5.00 scribed previously and does not resemble any other sequence and Ea = 125 kJ/mol for zebrafish). This functional adaptation stored in databases. meets the needs of the icefish transporter to work adequately be- The characterization of altered transport function in relation low 0 °C. A similar strategy is used by almost all cold-adapted to the unique COOH-terminal domain revealed that the deletion

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Fig. 4. Temperature dependence of chimeric rabbit–icefish PEPT1. (A) Schematic representation, (B)Q10 values, (C) Arrhenius plots, and (D) activation energy values (Ea) of wild-type and chimeric PEPT1 transporters. Experimental details are the same as for Fig. 3. a, difference vs. icefish PEPT1; b, difference vs. rabbit PEPT1 (one-way ANOVA/Bonferroni post hoc test).

of the whole set of repeated VDMSRKS units from the icefish additional module that causes adaptation to cold by decreasing protein results in loss of part of the cold-adapted phenotype. the Q10 and Ea values of the transport. The cytosolic VDMSRKS Maintaining a single VDMSRKS repeat in the COOH terminus is domain may influence protein flexibility by itself, by cooperating sufficient to keep full cold adaptation. This suggests that all the with other parts of the protein, or by interacting with the lipid different isoforms of PEPT1 transporters we identified in Ant- bilayer and/or with (an)other protein(s). In this respect, in the arctic fishes (i.e., the C. hamatus PEPT1 carrying a variable context of protein structure evolution, VDMSRKS clearly rep- number of VDMSRKS domains, and the PEPT1 proteins of the resents a rare case of emergence of a de novo domain (44, 45). other Antarctic fishes studied with only a single copy of this con- In conclusion, we identified a domain in the COOH terminus of served domain) are equally well adapted to operate at low tem- the PEPT1 transporter of the icefish C. hamatus that allows this fi peratures. Incorporating the ice sh VDMSRKS repeat region into integral transmembrane protein to operate at subzero temper- the COOH terminus of a warm-adapted (rabbit) PEPT1 trans- atures. The insertion of this additional unique amino acid stretch porter renders the protein cold adapted, showing that the transfer fi composed of seven amino acids contributes to cold adaptation, of this speci c module from the donor protein also transfers and the trait may be transferred to mammalian proteins, rendering a specific thermal trait to the acceptor protein. them cold adapted. This represents a unique strategy among those The temperature-dependent adaptive change observed in the already known in proteins from psychrophilic species (1, 2, 4, 5, primary amino acid sequence of icefish PEPT1 occurs in the 31). Moreover, it is unique that in a protein ortholog such a de COOH-terminal region beyond TMD XII. This region is poorly fi conserved among vertebrate PEPT1 orthologs. Moreover, this novo domain could be identi ed that carries a transferable func- region is not involved in the transport activity, as assessed by tional trait. In this respect, the VDMSRKS domain also may be computational, site-directed mutagenesis, chimeric, and other useful as a molecular tool for future studies including new approaches (36–43). Insertion of the VDMSRKS domain into this biotechnological applications. region does contribute to the cold adaptation of the protein. Very Materials and Methods little is known about the molecular changes that allow adaptation of Molecular Cloning. The molecular methods and primers used to clone the full- transmembrane transport proteins to work at low temperature, and fi it is assumed that the general principles that regulate cold adapta- length cDNA encoding for ice sh (C. hamatus) pept1, the genomic region covering the last two exons and intron of icefish pept1, and the cDNA tion in other proteins, such as cytoplasmic enzymes, also may apply fragments encoding for the COOH-terminal region of pept1 from T. bernacchii, to transmembrane proteins (16). As a common mechanism of ad- T. pennellii,andH. velifer are described in detail in SI Materials and Methods aptation to low temperatures in cytoplasmic enzymes, adaptive and Tables S3 and S4. changes occur in the sites in the amino acid sequence that influ- ence the conformational mobility during the catalytic cycle (3, 4). Site-Directed Mutagenesis and Chimeric Clones. A Site-directed Ligase- Although such a mechanism of adaptation may occur in icefish Independent Mutagenesis (SLIM) protocol (46) was used to generate the PEPT1, we demonstrate that a domain inserted in a region of the icefish pept1 mutants in the COOH-terminal region. A modified In-Fusion protein not directly involved in the catalytic events operates as an protocol (Clontech) was used to generate the rabbit–icefish chimeric cDNAs.

Rizzello et al. PNAS Early Edition | 5of6 Downloaded by guest on October 2, 2021 The molecular methods and primers used are described in detail in SI dependence of wild-type and mutant PEPT1 transport activity was assayed Materials and Methods and Table S5. over a temperature range of 280–300 K (7–27 °C). Temperature coefficient (Q10) and activation energy (Ea) were calculated for each transporter as in- RT-PCR and Real-Time PCR. Standard RT-PCR and real-time PCR protocols were dices of temperature dependence. Details on the methods are given in SI used. Primers and experimental conditions are provided in SI Materials and Materials and Methods. Methods and Table S6. ACKNOWLEDGMENTS. We thank Dr. A. Danieli for technical assistance and Dr. F. Pignatelli and Dr. L. Tagliaferro for real-time PCR analysis. This work X. laevis Oocytes and Electrophysiology. Wild-type or mutant pept1 cRNA was was supported by the Italian National Research Program in Antarctica, by injected into stage V/VI X. laevis oocytes, and two-electrode voltage clamp grants from the University of Salento (Fondi ex-60%), and in part by grants experiments were performed as described previously (28). Temperature from the Apulian Region (Codice Identificativo Progetto PS_070).

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