G Model

ARTICLE IN PRESS

PEP 69488 1–8

Peptides xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Peptides journal homepage: www.elsevier.com/locate/peptides

Hyperglycemic activity of the recombinant crustacean hyperglycemic hormone B1 isoform (CHH-B1) of the Pacific white shrimp Litopenaeus vannamei

1

2

3

Q1

4 5

Q2

6 7 8 9 10 11 12 13

Laura Camacho-Jiménez a , Edna Sánchez-Castrejón a , Elizabeth Ponce-Rivas a,∗ , b ˜ Ma. Enriqueta Munoz-Márquez , Manuel B. Aguilar c , Ana Denisse Re d , Fernando Díaz d a

Laboratorio de Biología Celular y Molecular, Departamento de Biotecnología Marina, Centro de Investigación Científica y Educación Superior de Ensenada (CICESE), Carretera Ensenada-Tijuana #3918, Ensenada, Baja California C.P. 22860, Mexico b Facultad de Ciencias Químicas e Ingeniería, Universidad Autónoma de Baja California (UABC), Av. Tecnológico s/n, Mesa de Otay, Tijuana, Baja California C.P. 22390, Mexico c Laboratorio de Neurofarmacología Marina, Instituto de Neurobiología, Universidad Nacional Autónoma de México (UNAM), Blvd. Juriquilla 3001, Juriquilla, Querétaro C.P. 76230, Mexico d Laboratorio de Ecofisiología de Organismos Acuáticos, Departamento de Biotecnología Marina, Centro de Investigación Científica y Educación Superior de Ensenada (CICESE), Carretera Ensenada-Tijuana #3918, Ensenada, Baja California C.P. 22860, Mexico

14

15 29

a r t i c l e

i n f o

a b s t r a c t

16 17 18 19 20 21

Article history: Received 6 March 2015 Received in revised form 22 May 2015 Accepted 26 May 2015 Available online xxx

22

28

Keywords: Crustacean hyperglycemic hormone Crustacean neurohormone Recombinant expression Dose–response hyperglycemic analysis Litopenaeus vannamei

30

Introduction

23 24 25 26 27

Crustacean hyperglycemic hormone (CHH) is the most abundant neuropeptide produced by the Xorgan/sinus gland (XO/SG) complex in the crustacean eyestalk. CHH plays a principal role in the control of glucose metabolism. The CHH-B1 isoform is produced in the eyestalk of Litopenaeus vannamei by alternative splicing of the chhB gene and its cDNA sequence has revealed that this isoform has a nonamidated C-terminal residue (CHH-like peptide). In this work, a recombinant CHH-B1 (rCHH-B1) with a sequence identical to the native hormone was expressed in the methylotrophic yeast Pichia pastoris X-33 and purified from the culture medium by RP-HPLC. The identity of the purified rCHH-B1 was confirmed by N-terminal sequencing and by using an anti-CHH-B1 polyclonal antibody. An in vivo assay showed that the hyperglycemic effect was dependant of the dosage of rCHH-B1, and the maximal hyperglycemic response was obtained with 250 pmol treatment. These results suggest that the amino acid sequence of the C-terminus and its correct structure are both important for the hyperglycemic activity of naturally occurring non-amidated CHH peptides, such as CHH-B1. CHH-B1 appears to be the first reported CHH-like peptide with significant hyperglycemic activity produced in the sinus gland of a penaeid shrimp. © 2015 Published by Elsevier Inc.

Q3 31 32 33 34 35 36

In crustaceans, many physiological processes are regulated by neuropeptides that belong to the crustacean hyperglycemic hormone (CHH) family, mainly produced by the X-organ/sinus gland (XO/SG) complex in the eyestalks. The most abundant neuropeptide produced by this complex is CHH, which is involved in the control of glucose levels in hemolymph [11]. However, multiple functions

Abbreviations: PAM, point accepted mutation; PBS, phosphate saline buffer; PCR, polymerase chain reaction; RP-HPLC, reverse-phase high performance liquid chromatography; SDS-PAGE, sodium dodecylsulfate-polyacrylamide gel electrophoresis; TCA, trichloroacetic acid; TFA, trifluoroacetic acid. ∗ Corresponding author. Tel.: +52 646 1750500x27162. E-mail address: [email protected] (E. Ponce-Rivas).

have been reported for CHH, including regulation of lipid metabolism [31], molting [6,7], osmoregulation [33,36], reproduction [9,42] and stress response [1]. The multiple activities have been explained as being due to the existence of CHH isoforms that may originate by various mechanisms, including the transcription of multiple copies of genes [13], alternative splicing of genes [10], or by post-translational processing such as L to D isomerization [34,35], and N-terminal blocking [5]. The CHH genes are classified into two types according their exon–intron organization: the type I genes that have 4 exons and 3 introns, and the type II genes that have 3 exons and 2 introns. Most of the type I genes described so far encode two transcripts that, in most cases, share the signal peptide sequence, the CHH precursor-related peptide (CPRP) and the N-terminal region (exons I and II), but differ in the C-terminal region, which can be encoded by exon III or exon IV [4,10]. These alternative splicing isoforms

http://dx.doi.org/10.1016/j.peptides.2015.05.014 0196-9781/© 2015 Published by Elsevier Inc.

Please cite this article in press as: Camacho-Jiménez L, et al. Hyperglycemic activity of the recombinant crustacean hyperglycemic hormone B1 isoform (CHH-B1) of the Pacific white shrimp Litopenaeus vannamei. Peptides (2015), http://dx.doi.org/10.1016/j.peptides.2015.05.014

37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52

G Model PEP 69488 1–8

L. Camacho-Jiménez et al. / Peptides xxx (2015) xxx–xxx

2 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94

95

96

97 98 99 100 101 102 103 104 105 106 107 108

109

110 111 112

ARTICLE IN PRESS

have been found in various crustaceans, including Carcinus maenas [10], Macrobrachium rosenbergii [3], Pachygrapsus marmoratus, Potamon ibericum [38], Gecarcinus lateralis [22], Callinectes sapidus [8,53], Scylla olivacea [41], Pandalopsis japonica [16], Procambarus clarkii [19,47,48,50], Portunus trituberculatus [49], and Litopenaeus vannamei [21]. Isoforms originating by alternative splicing have different expression patterns, where at least one transcript has been detected in extra-eyestalk tissues. Interestingly, in all cases only one of the isoforms has an amide moiety at the C-terminus, which is characteristic of CHH neuropeptides, while the other isoform has a free C-terminus (CHH-like or CHH-L) [23,46]. In the Pacific white shrimp L. vannamei, three different CHH isoforms have been cloned and sequenced: CHH-A (GenBank accession no. AY434016), CHH-B1 (GenBank accession no. AY167045), and CHH-B2 (GenBank accession no. AY167046). The CHH-B1 and CHH-B2 isoforms are both produced in the eyestalk of L. vannamei by alternative splicing of the chhB gene (formerly referred as molt-inhibiting hormone, mih) [21]. The sequence analysis of mRNA transcripts indicated that the CHH-B1 isoform is a CHH-L neuropeptide, whereas CHH-B2 has a processing signal (GK) for ␣-amidation of the C-terminal valine amino acid residue (CHH peptide). CHH-B1 expression has been shown to be higher than that for CHH-B2 in eyestalks and the gene expression of these neurohormones seems to be highly dependent on the environmental salinity and temperature [21]. CHH-B1 has also been detected in tissues other than the eyestalk [37]. Those findings suggest that CHH-B1 plays an important physiological role in L. vannamei, and may also have some of the additional functions attributed to CHHs, which have not been studied previously in this species. CHH-B1 has been cloned previously and expressed in the methylotrophic yeast Pichia pastoris, but with 28 additional amino acids at the C-terminus (c-myc/polyhistidine tags) that allowed the easy identification and purification of the neurohormone with commercial antibodies and metal affinity chromatography, respectively. Even though the recombinant peptide showed hyperglycemic activity in vivo, it did not show molt-inhibiting activity [30]. In the present work, we report the cloning in P. pastoris, expression and purification of the L. vannamei recombinant CHH-B1 isoform with a free C-terminus (rCHH-B1) and identical to the native neurohormone, as well as an examination of its hyperglycemic activity using a dose–response bioassay with bilaterally ablated shrimp. Materials and methods Animals The L. vannamei shrimp used in these experiments were acquired as post-larvae from the commercial farm Acuacultura Mahr in La Paz, México. The post-larvae were grown until they reached juvenile stage in tanks filled with aerated seawater at 35‰ and 28 ± 1 ◦ C. For the hyperglycemia assay, 200 sub-adult shrimp were transferred to individual containers (3.5 L) submerged in tanks with aerated seawater at 35‰ and 26 ± 1 ◦ C. The animals were fed twice a day with a commercial diet supplemented with 4% squid meal to enrich protein content in the food. Daily, debris and feces were removed, and the seawater was completely exchanged. The shrimp were acclimated to these conditions for 10 days before starting the bioassay. Construction of the expression vector for rCHH-B1 Eyestalks from juvenile shrimp were dissected for total RNA extraction. After removal of the cuticle and non-neural tissues, eyestalks were homogenized with Tri Reagent (Sigma–Aldrich,

Saint Louis, MO, USA) and RNA was isolated according to the manufacturer’s instructions. Total RNA (1 ␮g) was treated with DNAse I, Amplification Grade (Invitrogen, Life Technologies, Carlsbad, CA, USA) and first strand cDNA was synthesized using SuperScript III Reverse Transcriptase and an Oligo (dT)20 primer (Invitrogen, Life Technologies), according to the manufacturer’s protocol. The chh-B1 transcript was amplified by PCR using the forward primer 5 -TTGAGAAGCTGCTGTCGTCCT-3 and the reverse primer 5 -CTTGTTTCCTCCACATTAGCG-3 [21]. The PCR reaction (50 ␮L) consisted of 1X Green GoTaq® Flexi Buffer (Promega, Madison, WI, USA), 3 mM MgCl2 , 0.2 mM of each dNTP, 0.2 ␮M of each primer, 1.25 U of GoTaq® DNA Polymerase, and 2.0 ␮L of the reverse transcription reaction mix. The PCR was carried out at 95 ◦ C for 2 min, followed by 30 cycles of 95 ◦ C for 30 s, 60 ◦ C for 45 s and 72 ◦ C for 45 s, and a final extension step at 72 ◦ C for 5 min. The amplified PCR product of about 500 bp was purified using a PureLinkTM PCR Purification kit (Invitrogen, Life Technologies), and used as template in a second PCR reaction. For cloning the mature chh-B1 transcript into the pPICZ␣A vector (Invitrogen, Life Technologies), specific primers were designed. The forward primer 5 -CCGCTCGAGAAAAGAGACACCTTCGACCACTCCTGCAAGG-3 includes a XhoI site (underlined), and deletes the Ste13 signal cleavage site (two E–A repeats) between the ␣-factor secretion signal sequence and the mature chh-B1 transcript (bold). The reverse primer 5 -GCTCTAGATTAGGGATAGCGCAGAAA-3 includes a XbaI site (underlined) and stop codon (bold). The mature chh-B1 transcript was amplified by PCR (100 ␮L) using the cDNA PCR fragment (1.0 ␮L) previously purified. PCR mix and settings were identical to those described above. The purified chh-B1 fragment and pPICZ␣A vector were digested with XhoI and XbaI restriction enzymes (Promega) and ligated with T4 DNA ligase (New England BioLabs, Ipswich, MA, USA) according to the manufacturer’s protocol. The ligation product was used to transform Escherichia coli DH5␣ cells by electroporation in a Bio-Rad Micropulser (2.5 kV, 1 pulse, ∼5 ms). Transformant colonies were screened for zeocin (Invitrogen, Life Technologies) resistance and evaluated by PCR using 5 AOX1 and 3 AOX1 primers (5 AOX1, 5 -GACTGGTTCCAATTGACAAGC-3 ; 3 AOX1, 5 -GCAAATGGCATTCTGACATCC-3 ). The plasmid vector construction was verified by sequencing (SeqxCel, San Diego, CA, USA). The pPICZ␣A-CHH-B1a vector was linearized with BstXI (New England BioLabs) for integration into the P. pastoris X-33cell genome by electroporation (2.0 kV, 1 pulse, ∼5 ms), as described in the manual (version G) of the EasySelect Pichia Expression Kit (Invitrogen, Life Technologies). The transformants were screened for zeocin resistance on YPDS plates (1% yeast extract, 2% peptone, 2% dextrose, 1 M sorbitol, 2% agar, 100 ␮g/mL zeocin) and integration of the construct into the yeast genome was evaluated by PCR and sequence analysis. Sequence analysis The amino acid sequence of mature CHH-B1 was aligned against CHH and CHH-like (CHH-L) isoforms originated by alternative splicing. The sequences were obtained from the NCBI GenBank database (http://ncbi.nlm.nih.gov). The multiple sequence alignment was performed with the software BioEdit 7.25 [15], using standard parameters such as the PAM250 residue weight table, and gap penalty of 10. Expression of rCHH-B1 The expression of rCHH-B1 was induced according to the method described by Sánchez-Castrejón et al. [30] with some modifications. A colony of P. pastoris X-33 containing pPicZ␣A-CHH-B1a inserted into its genome was grown in 3 mL of YPD medium (1% yeast extract, 2% peptone, 2% dextrose, 100 ␮g/mL zeocin) for 18 h

Please cite this article in press as: Camacho-Jiménez L, et al. Hyperglycemic activity of the recombinant crustacean hyperglycemic hormone B1 isoform (CHH-B1) of the Pacific white shrimp Litopenaeus vannamei. Peptides (2015), http://dx.doi.org/10.1016/j.peptides.2015.05.014

113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160

161

162 163 164 165 166 167 168

169

170 171 172 173 174

G Model PEP 69488 1–8

ARTICLE IN PRESS L. Camacho-Jiménez et al. / Peptides xxx (2015) xxx–xxx

195

at 30 ◦ C and 200 rpm. The culture (1 mL) was transferred to 500 mL of BMGY medium (1% yeast extract, 2% peptone, 0.67% yeast nitrogen base (YNB), 4 ␮g/m LD-biotin, 100 mM potassium phosphate buffer pH 6.0, 1% glycerol), and incubated under the same conditions until the cell density reach an OD600 of ∼4. The inoculum was concentrated 5 times by centrifugation (2500 × g, 5 min) and the pellets were resuspended in 20 mL of BMMY medium (1% yeast extract, 2% peptone, 0.67% YNB, 4 ␮g/mL D-biotin, 100 mM potassium buffer pH 6.0), with different final concentrations of methanol (0, 1, and 2%, v/v). Fresh methanol was added every 12 h to sustain the induction for 48 h. Daily, 1 mL of culture was centrifuged (13,000 × g, 5 min) and the supernatant was recovered and stored at −20 ◦ C for further analysis. The non-transformed X-33 strain was used as negative control for expression. The expression of the recombinant peptide was analyzed by electrophoresis. Proteins in the supernatant samples were precipitated by adding trichloroacetic acid (TCA) to a final concentration of 15% (v/v), and resuspended in Laemmli sample buffer [20]. The proteins in samples were electrophoretically separated by Tricine SDS-PAGE (12.5%) [32], and stained with Coomassie brilliant blue (R-250).

196

Immunodetection of rCHH-B1

175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194

218

The presence of the heterologous peptide was confirmed by a Western blot assay. The proteins separated on the gel were transferred to a 0.45 ␮m Trans-Blot Transfer Medium (Bio-Rad, Hercules, CA, USA) nitrocellulose membrane by the semidry blotting method [12] using a Semi-Dry Electroblotter System (Apollo, Continental Lab Products, San Diego, CA, USA). For the immunodetection, the membrane was blocked overnight at 4 ◦ C in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2 HPO4 , 1.8 mM KH2 PO4 , 0.05%, pH 7.4) with 0.05% Tween-20 (PBST), and 5% non-fat milk. Subsequently, the membrane was washed 3 times for 5 min with PBST and incubated with a primary rabbit anti-CHH-B polyclonal affinity antibody (GeneScript, Piscataway, NJ, USA) in PBST with 1% non-fat milk (diluted 1:500) for 4 h at room temperature. The antiCHH-B polyclonal antibody was produced using a KLH-conjugated, synthetic peptide designed from the N-terminal sequence of the L. vannamei neurohormones CHH-B1 and CHH-B2. Subsequently, the membrane was washed and incubated with a secondary goat anti-rabbit IgG (whole molecule)-peroxidase monoclonal antibody (Sigma–Aldrich) (diluted 1:5000) under the same conditions. Finally, the membrane was washed and the signal was developed with 3,3 ,5,5 -Tetramethylbenzidine (TMB) solution (1-Step TMBBlotting; Pierce, Rockford, IL, USA).

219

Purification of rCHH-B1

197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217

220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236

For rCHH-B1 purification, the protein expression was performed in 200 mL BMMY culture medium under the selected expression conditions. The rCHH-B1 peptide was partially purified and concentrated from the supernatant by precipitation with 50% ammonium sulfate and then dialyzed against PBS using a 3500 MWCO SlideA-Lyzer Dialysis Cassette (Thermo Scientific, Rockford, IL, USA). Final purification was achieved by RP-HPLC using an Agilent 1100 system (Agilent Technologies, Wilmington, DE, USA) equipped with a multiple wavelength detector. Separation was performed on a TSKgel Octadecyl-4PW analytical column (4.6 mm × 150 mm, Tosoh, Tokyo, Japan). The column was equilibrated for 5 min with 0.12% TFA (solvent A) and then eluted with a gradient of 0–55% acetonitrile with 0.1% TFA (solvent B) at a flow rate of 1 mL/min, during 55 min. The absorbance of the eluted material was monitored at 280 nm. Fractions of 5 min between 20 and 45 min retention times were collected, lyophilized and dissolved in PBS. The presence of the recombinant peptide in the collected fractions was evaluated

3

by a Dot blot assay using the anti-CHH-B1 polyclonal antibody as described in Section “Immunodetection of rCHH-B1”. The fractions that contained rCHH-B1 were pooled and re-analyzed by TricineSDS-PAGE electrophoresis and immunodetected by Western blot. The concentration of purified rCHH-B1 was determined with the BCA Protein Assay Kit (Pierce). To determine the N-terminal sequence of the two bands identified by the anti-CHH-B antibody, the bands were separated by Tricine-SDS-PAGE as previously described, blotted onto 0.2 ␮m Sequi-Blot PVDF membrane (Bio-Rad), and excised from the membrane. N-terminal sequencing was performed by Edman degradation using a Procise 491 Protein Sequencing System (Applied Biosystems, Foster City, CA, USA). Bioassay for hyperglycemic activity in vivo A biological assay was done using 56 shrimp (12.9 ± 2.4 g) acclimated as described in Section “Animals”. The intermolt stage of each shrimp was estimated as the half of the interval between two consecutive molts registered by the observation of exuviae in each individual container. In the first registered intermolt, one eyestalk was ablated by cutting and cauterization. In order to allow the animals to recover from the injury, removal of the second eyestalk was delayed until the next intermolt. Bilaterally eyestalk-ablated shrimp in intermolt were deprived of food for 24 h before the assay. To examine the dose–response relationship of rCHH-B1 activity, different doses (5, 10, 50, 100, 250, 500 and 1000 pmol) were prepared by dissolving rCHH-B1 in 50 ␮L of PBS and then injected into shrimp through the arthrodial membrane of the fifth walking leg, with a 1 mL sterile hypodermic syringe (31 G). For negative and positive controls, 50 ␮L of PBS and a pair of sinus glands dissolved in PBS were injected, respectively. Experimental treatments and control groups included 6–7 organisms. Hemolymph was collected (50 ␮L) from the dorsal side of the first abdominal segment, 1 h after the injections, with a 1 mL sterile hypodermic syringe (27 G) and diluted with 100 ␮L of shrimp salt solution (SSS) (450 mM NaCl, 10 mM KCl, 10 mM EDTA·Na2 , 10 mM HEPES, pH 7.3, 850 mOsm/kg) as anticoagulant [44]. The plasma fraction of the hemolymph was separated by centrifugation (855 × g, 5 min at 4 ◦ C) and stored at −80 ◦ C until analysis. Glucose levels in each sample were analyzed in triplicate using the glucose oxidase method with a Liquid Glucose Reagent Set (Pointe Scientific, Canton, MI, USA). Statistical analysis Glucose concentration results were analyzed by the Kruskal–Wallis non-parametric ANOVA test and differences among groups were determined by Dunn’s multiple comparison post hoc test using SigmaPlot 10.0 (Systat Software). The value p < 0.05 was considered to be statistically significant and values were expressed as median ± 95% confidence interval.

237 238 239 240 241 242 243 244 245 246 247 248 249

250

251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276

277

278 279 280 281 282 283

Results

284

Construction of the expression vector for recombinant rCHH-B1

285

The mature chh-B1 transcript was cloned into the pPICZ␣A vector producing the pPICZ␣A-CHH-B1a vector (Fig. 1). Sequencing of vector pPICZ␣A-CHH-B1a showed no mutations and the reading frame with the ␣-factor protein secretion signal was corroborated. Four of the 5 P. pastoris transformants screened by PCR contained the expected 750 bp amplicon. Sequencing analysis of the transformants confirmed the integration of the mature chh-B1 sequence into the genome of P. pastoris X-33 producing the X-33 CHH-B1a strain. This strain expresses the 73 aa rCHH-B1 peptide, with a theoretical mass in reduced form of 8882.16 Da.

Please cite this article in press as: Camacho-Jiménez L, et al. Hyperglycemic activity of the recombinant crustacean hyperglycemic hormone B1 isoform (CHH-B1) of the Pacific white shrimp Litopenaeus vannamei. Peptides (2015), http://dx.doi.org/10.1016/j.peptides.2015.05.014

286 287 288 289 290 291 292 293 294 295

G Model PEP 69488 1–8

ARTICLE IN PRESS L. Camacho-Jiménez et al. / Peptides xxx (2015) xxx–xxx

4

Fig. 1. Diagram of the pPICZ␣A-CHH-B1a construction.

296

297 298 299 300 301 302 303 304 305 306 307 308 309 310

Sequence analysis

Expression and identification of rCHH-B1

The multiple sequence alignment of CHH-B1 with other CHH/CHH-L mature peptides showed that most of the consensus sequence is located within the first 40 amino acids at the N-terminal region. The main differences were observed at the C-terminal region of the CHH-L peptides (Fig. 2). The CHH-B1 neuropeptide showed the highest identity with Lv-CHH-B2 (64.8%), followed by P. japonica Pj-CHH1PO (54.6%) and other CHH-L neuropeptides such as P. clarkii Prc-CHH1-L (54%) and M. rosenbergii Mar-CHH-L (52%). As can be seen in Fig. 2, unlike the CHH isoforms, the CHHL neuropeptides that have been analyzed so far have not shown hyperglycemic activity except for the blue crab CsPO-CHH, which showed hyperglycemic activity using high doses of the recombinant hormone (150–200 pmol) [18], and the Liv-CHH-B1 from this work.

The expression of rCHH-B1 in a secreted form was analyzed in the culture supernatant. The analysis by Tricine-SDS-PAGE demonstrated the production of a band with a relative molecular weight of ∼10 kDa, which agrees well with the expected size (8882.16 Da) for rCHH-B1 (reduced) in all the cultures with methanol. A low level of additional secreted proteins was also observed (Fig. 3A). The expected band had a similar intensity in the samples from the cultures with 1% methanol (24 h) and 2% methanol (24–48 h). An additional band of ∼15 kDa in minor abundance was also observed. Both bands were immunodetected using the anti-CHH-B antibody (Fig. 3B). Bands of ∼10 kDa were not detected in control samples. Since the Western blot analysis of culture supernatants showed that the expression of rCHH-B1 was similar among the induction conditions analyzed, the condition of 2% methanol for 24 h was selected

Fig. 2. Multiple alignment of CHH/CHH-L mature peptide sequences. Abbreviations and GenBank accession numbers: Pam, P. marmoratus (CHH-B XO, AAO27805; CHH-B PO, AAO27806) [38]; Pt, P. trituberculatus (CHH-1, ACB46189; CHH-2, AIZ94611) [49]; Cs, C. sapidus (ES-CHH (CHH-1), AAS45136; CsPO-CHH (CsCHH-2, ABG67921, ABC61678) [8,18,53]; Gel, G. lateralis (EG-CHH-A, ABF48652; Po-CHH, ABF58091) [22,52]; Cam, C. maenas (SG-CHH, AAG29442; PO-CHH, AAG29435) [10]; Sco, S. olivacea (CHH, AAQ75760; CHH-L, ABP88270) [2,41]; Poi, P. ibericum (CHHXO, ABA70560; CHHPO, ABA70561) [38]; Mar, M. rosenbergii (CHH, AAF29534; CHH-L, AF372657) [3,29]; Prc, P. clarkii (CHH1, BAA89003; CHH1-L, AAL79193; CHH2, AFV95082; CHH2-L, AFV95078) [19,48]; Pj, P. japonica (CHH1ES, AFG16933; CHH1PO, AFG16932) [16]; Lv, L. vannamei (CHH-B2, AAN86057; CHH-B1, AY167045) [21,30]; Hyperglycemic activity tested in vivo: N, using native neuropeptides; R, recombinant neuropeptides; (+), with activity; (−) without activity; aa, number of amino acids in sequences; % I.D., identity percentage relative to the CHH-B1 sequence. CHH, CHH isoforms; CHH-L, CHH-like isoforms. *, indicates the consensus sequence (100% identity).

Please cite this article in press as: Camacho-Jiménez L, et al. Hyperglycemic activity of the recombinant crustacean hyperglycemic hormone B1 isoform (CHH-B1) of the Pacific white shrimp Litopenaeus vannamei. Peptides (2015), http://dx.doi.org/10.1016/j.peptides.2015.05.014

311

312 313 314 315 316 317 318 319 320 321 322 323 324 325

G Model PEP 69488 1–8

ARTICLE IN PRESS L. Camacho-Jiménez et al. / Peptides xxx (2015) xxx–xxx

5

Fig. 3. Expression analysis of rCHH-B1. (A) Tricine-SDS-PAGE. M, Precision Plus Protein Unstained Standards (Bio-Rad); 1–3, 0% methanol for 0, 24 and 48 h, respectively; 4–6, 1% methanol for 0, 24 and 48 h, respectively; 7–9, 2% methanol for 0, 24 and 48 h, respectively. (B) Western blot. M, Precision Plus Protein All Blue Standards (Bio-Rad); 1, 1% methanol for 0 h; 2–3, 1% methanol for 24 and 48 h, respectively; 4–5; 2% methanol for 24 and 48 h, respectively; 6, 0% methanol for 48 h; 7–8, X-33 strain without vector with 0% and 2% methanol, for 24 h, respectively. The arrows indicate the bands corresponding to rCHH-B1.

327

based on the shortest time for expressing a significant amount of recombinant neurohormone.

328

Purification of rCHH-B1

326

329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348

Because the most defined peaks detected by the RP-HPLC had a retention time between 20 and 45 min, 5 fractions were collected in that interval (F1–F5). The higher peaks in the chromatogram were observed for F3 (30–35 min) and F4 (40–45 min) (Fig. 4). Dot blot assay of the 5 fractions using the anti-CHH-B antibody confirmed the presence of the rCHH-B1 peptide mainly in F3 and F4. These fractions were pooled and re-analyzed. The expected rCHH-B1 band of ∼10 kDa was observed on Tricine-SDS-PAGE (left inset to Fig. 4) and detected by the anti-CHH-B antibody (right inset to Fig. 4). The additional band of ∼15 kDa was also observed in the Western blot analysis, but less intense than the ∼10 kDa band. The absence of high molecular weight bands in pooled fractions confirmed the removal of all additional proteins during the stages of ammonium sulfate precipitation and chromatography. The final yield of the purified rCHH-B1 was 7 mg/L of culture medium. The N-terminal sequence analysis of the ∼10 kDa band from the purified rCHH-B1 sample revealed that the first six amino acids (DTFDHS) matched with the expected deduced sequence for mature rCHH-B1, indicating the correct processing of the ␣-factor secretion signal. The N-terminal amino acid sequence obtained for

the ∼15 kDa band revealed the presence of rCHH-B1, and suggests the formation of non-covalent aggregates (probably, dimers) of denatured rCHH-B1 molecules. In addition, small proportions of the sequence GLOGTA (where O is 4-hydroxy-proline) were detected in both the ∼10 kDa band (2.8%) and the ∼15 kDa band (14.9%); this sequence corresponds to fragments of collagen, which is a component of the peptone included in the YPD, BMGY, and BMMY culture media employed for inducing the expression of rCHH-B1. Bioassay for hyperglycemic activity in vivo The hyperglycemic activity of rCHH-B1 was assessed by an in vivo dose–response assay. The mortality rate during the experiment was ∼70%, possibly due to the stress caused by the bilateral eyestalk ablation procedure. As can be seen in Fig. 5, injection of 5 pmol of rCHH-B1 increased hemolymph glucose levels by 4.3 ± 0.29 mg/dL, which was not significantly different from the level elicited by PBS injection (2.72 ± 0.36 mg/dL). Injection of 10 pmol or more of the recombinant hormone increased the hemolymph glucose levels significantly. However, glycemia did not increase significantly with doses above 250 pmol. Thus, the minimal and maximum hyperglycemic activity promoted by the injection of rCHH-B1 into juvenile L. vannamei shrimps were 13.58 ± 1.36 mg/dL (with 10 pmol) and 24.62 ± 1.04 mg/dL (with 250 pmol), respectively. Discussion

Fig. 4. RP-HPLC profile of rCHH-B1 purification. F1–F5, fractions collected from the column and their respective Dot blot analysis; + synthetic peptide as positive control, −, PBS as negative control. The transverse line indicates the concentration of solvent B; C, Tricine-SDS-PAGE analysis of the pooled fractions after the RP-HPLC purification step; WB, Western blot analysis of purified rCHH-B1.

In crustaceans, CHH is a key neurohormone that performs several important physiological functions during their development and life cycle. Although the principal activity of CHH has been related to carbohydrate metabolism, recent studies have discovered that CHH has multiple roles [7,31,33,42]. The presence of multiple structural isoforms of CHH has suggested that they have different physiological functions; thus, the expression of active recombinant CHHs is critical for studying their role in crustacean metabolism and physiology. In order to clarify the dose–response relationship of the recombinant isoform CHH-B1 of L. vannamei and its hyperglycemic activity, CHH-B1 was expressed in P. pastoris as a secreted peptide with a sequence identical to that of the native hormone, and expected to have the structure and activity of the native neurohormone. Even though the recombinant CHH-B1 peptide has no additional tags that aid its purification, it was successfully purified by RP-HPLC. This purification strategy has been used before to obtain other bioactive recombinant CHH-family peptides expressed in P. pastoris, for example Pem-CHH1-3, Pem-MIH1 and Pem-GIH from Penaeus monodon [39,40,43,51], and Pej-SGPIII from Penaeus japonicus [28]. The N-terminal sequence analysis

Please cite this article in press as: Camacho-Jiménez L, et al. Hyperglycemic activity of the recombinant crustacean hyperglycemic hormone B1 isoform (CHH-B1) of the Pacific white shrimp Litopenaeus vannamei. Peptides (2015), http://dx.doi.org/10.1016/j.peptides.2015.05.014

349 350 351 352 353 354 355 356

357

358 359 360 361 362 363 364 365 366 367 368 369 370 371

372

373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393

G Model PEP 69488 1–8 6

ARTICLE IN PRESS L. Camacho-Jiménez et al. / Peptides xxx (2015) xxx–xxx

Fig. 5. Hyperglycemic activity of rCHH-B1. SG, sinus gland extracts; −, PBS as negative control (right panel). Results from rCHH-B1 doses (5, 10, 50, 100, 250, 500 and 1000 pmol) (left panel) and controls are plotted as median ± 95% confidence interval. Asterisk indicates significant differences from negative control (p < 0.05).

394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436

and the development of an anti-CHH-B polyclonal antibody allowed confirmation of the identity of the rCHH-B1. At the protein level, the consensus appears to be that the eyestalk predominantly expresses CHH isoforms with C-terminal amidation that show significant induction of hyperglycemia, suppression of ecdysteroid synthesis, or both, whereas the extraeyestalk tissues predominantly express CHH-L isoforms that have a free C-terminus and have generally been reported not to have hyperglycemic activity [2,10,29]. CHH-B1 and CHH-B2 isoforms are alternatively spliced products highly expressed in the eyestalk of L. vannamei [21]. Tiu et al. [37] have reported that LvITP, which has a 98.3% identity with the CHH-B1 sequence, is also transcribed in other non-eyestalk nervous tissues (such as brain, thoracic ganglion and ventral nerve cord), the epidermis, gill and gut. Interestingly, we found that CHH-B1 has hyperglycemic activity even though the sequence of the native CHH-B1 suggests that it has a free C-terminus. Our present results confirmed the hyperglycemic activity that was previously observed using a CHH-B1 recombinant version having 26 additional amino acids at the C-terminal end, constructed previously by our group using unilaterally ablated shrimp [30]. Differences in the hyperglycemic activity of CHH/CHH-L isoforms in crustaceans have been related to the N-terminal portion of the peptides [14,25,48]. Based on point and deletion mutants, it has also been suggested that the high degree of conservation of the six cysteines is related to the functional folding of the recombinant hormones and the hyperglycemic function [25]. The C-terminus of mature CHH peptides has been considered significant for hyperglycemic activity, but this is not yet fully understood. Some authors have observed that the presence of additional Cterminal tags could interfere with the hyperglycemic activity [52], as well as the generation of point and deletion mutants at the C-terminus [25], and forms truncated at the C-terminus lacked hyperglycemic activity [24]. In particular, C-terminal amidation has been considered to be critical for the hyperglycemic activity of alternatively spliced CHH isoforms that are naturally amidated, since differences in activity and in the conformation of the Cterminal region have been observed between C-terminal amidated and non-amidated recombinant peptides [17,26,27], and several authors have found lack of activity in some extra-eyestalk CHHL isoforms having non-amidated C-terminal residues. However, despite isoforms originating by alternative splicing (CHH/CHH-L) share an identical N-terminal sequence (residues 1–40), they differ

considerably in the remaining sequence. This suggests that the lack of activity of CHH-L peptides, like CHH-B1, may not be due only to the absence of an amide moiety in the C-terminal residue, but also to the amino acid sequence of the C-terminal region [2,10,29]. Another important point to consider is that in order to obtain biologically active CHH peptides the refolding reaction is required for correct arrangement of disulfide bonds in recombinant hormones produced from E. coli expression systems [2,17,29]. Recombinant neuropeptides may not be properly formed, being partially degraded or having improper folding as compared with the native hormone. There may be an incorrect arrangement of intramolecular disulfide bridges or changes in the ␣-helical content. In some such cases it has been necessary to inject high doses of recombinant CHHs (micrograms per animal) to induce a hyperglycemic response [14,18,45]. Accordingly, the correct conformation of the recombinant hormone, and the dosage used in the hyperglycemic bioassays, are crucial for properly assessing the activity of the hormone. In this sense, the use of dose–response curves could help in the correct characterization of the CHHs. The hyperglycemic activity obtained using 10 or 100 pmol of the free C-terminus rCHH-B1, expressed in P. pastoris, was about 3 times higher in comparison with the activity obtained using the same concentration of the recombinant P. japonicus CHHs with a C-terminal amide moiety such as the rPej-SGP-I-amide expressed in E. coli [17] and the rPej-SGP-III-amide expressed in P. pastoris [28]. Additionally, the effect of rCHH-B1was 2 times higher compared with the rPej-SGP-VII-amide produced in E. coli [27]. These results suggest that even though the C-terminal amide moiety of naturally amidated CHHs is significant in conferring high hyperglycemic activity, the amino acid sequence of the C-terminus of the neurohormones and its correct structure are important not only for the hyperglycemic activity observed in some non-amidated versions of recombinant CHH isoforms that are naturally amidated [14,17,26,27], but also for the activity observed in the naturally occurring non-amidated isoforms like CHH-B1. In conclusion, to our knowledge, CHH-B1 is the first neuropeptide identified in penaeid shrimp that is produced in the sinus gland as a splicing isoform with a free C-terminus encoded by a type I CHH gene. The rCHH-B1 neurohormone from L. vannamei, produced as a free C-terminal neurohormone, displayed dosedependent hyperglycemic activity in a bioassay with bilaterally ablated shrimp, suggesting that even though the C-terminal ␣amidation could be important for hyperglycemic activity of some

Please cite this article in press as: Camacho-Jiménez L, et al. Hyperglycemic activity of the recombinant crustacean hyperglycemic hormone B1 isoform (CHH-B1) of the Pacific white shrimp Litopenaeus vannamei. Peptides (2015), http://dx.doi.org/10.1016/j.peptides.2015.05.014

437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479

G Model PEP 69488 1–8

ARTICLE IN PRESS L. Camacho-Jiménez et al. / Peptides xxx (2015) xxx–xxx

480 481 482 483 484 485 486 487 488 489 490 491 492 493 494

495

CHH neuropeptides, it is not a requisite for hyperglycemic activity of CHHs with a free C-terminus such as CHH-B1. Additional structure–function studies need to be done in order to have a better understanding of the role of CHHs on the regulation of hemolymph glucose levels in crustaceans. Since the CHH-B1 transcript is expressed in different tissues, and the expression pattern is altered by environmental conditions such as salinity and temperature, it will be important to explore the role of this isoform in other physiological processes related to lipid metabolism, osmoregulation, molting/growth, stress response, and reproduction in order to understand completely its relevance during the life cycle of the species. The successful synthesis and purification of a recombinant CHH-B1 identical to the native hormone will be useful for conducting studies on the diverse physiological roles of the members of the CHH family of L. vannamei. Acknowledgements

We thank Dr. John van der Meer for his helpful suggestions and his valuable help in polishing the English in the manuscript. This 497 Q4 work was funded by the National Council for Science and Technol498 ogy of Mexico (grant CB2009-133958-Z). 499 496

500

501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552

References [1] Chang ES. Stressed-out lobsters: crustacean hyperglycemic hormone and stress proteins. Integr Comp Biol 2005;45:43–50. [2] Chang C, Tsai K, Hsiao N, Chang C, Lin C, Watson RD, et al. Structural and functional comparisons and production of recombinant hyperglycemic hormone (CHH) and CHH-like peptides from mud crab Scylla olivacea. Gen Comp Endocrinol 2010;167:68–76. [3] Chen S, Lin C, Kuo CM. Cloning of two crustacean hyperglycemic hormone isoforms in freshwater gian prawn (Macrobrachium rosenbergii): evidence of alternative splicing. Mar Biotechnol 2004;6:83–94. [4] Chen S, Lin C, Kuo CM. In silico analysis of crustacean hyperglycemic hormone family. Mar Biotechnol 2005;7:193–206. [5] Chung JS, Webster SG. Does the N-terminal pyroglutamate residue have any physiological significance for crab hyperglycemic neuropeptides? Eur J Biochem 1996;240:359–64. [6] Chung JS, Dirksen H, Webster SG. A remarkable, precisely timed release of hyperglycemic hormone from endocrine cells in the gut is associated with ecdysis in the crab Carcinus maenas. PNAS 1999;96(23):13103–7. [7] Chung JS, Webster SG. Dynamics of in vivo release of molt-inhibiting hormone and crustacean hyperglycemic hormone in the shore crab, Carcinus maenas. Endocrinology 2005;146(12):5545–51. [8] Chung JS, Zmora N. Functional studies of crustacean hyperglycemic hormones (CHHs) of the blue crab, Callinectes sapidus – the expression and release of CHH in eyestalk and pericardial organ in response to environmental stress. FEBS J 2008;275:693–704. [9] De Kleijn DPV, Janssen KPC, Waddy SL, Hegeman R, Lai WY, Martens GJM. Expression of the crustacean hyperglycaemic hormones and the gonadinhibiting hormone during the reproductive cycle of the female American lobster Homarus americanus. J Endocrinol 1998;156:291–8. [10] Dircksen H, Böcking D, Heyn U, Mandel C, Chung JS, Baggerman G, et al. Crustacean hyperglycemic hormone (CHH)-like peptides and CHH-precursor related peptides from pericardial organ neurosecretory cells in the shore crab. Carcinus maenas, are putatively spliced and modified products of multiple genes. Biochem J 2001;356:159–70. [11] Fanjul-Moles ML. Biochemical and functional aspects of crustacean hyperglycemic hormone in decapod crustaceans: review and update. Comp Biochem Physiol C 2006;142:51–9. [12] Gallagher S, Winston SE, Fuller SA, Hurrell JGR. Immunoblotting and immunodetection. In: Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, et al., editors. Current protocols in molecular biology. New York: John Wiley & Sons Inc.; 2003, 10.8.1-24. [13] Gu P, Chan S. The shrimp hyperglycemic hormone-like neuropeptide is encoded by multiple copies of genes arranged in a cluster. FEBS Lett 1998;441:397–403. [14] Gu P, Yu KL, Chan S. Molecular characterization of an additional shrimp hyperglycemic hormone: cDNA cloning, gene organization, expression and biological assay of recombinant proteins. FEBS Lett 2000;472:122–8. [15] Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser 1999;41:95–8. [16] Jeon J, Kim B, Lee JH, Kim HJ, Kang C, Mykles DL, et al. Two type I crustacean hyperglycemic hormone (CHH) genes in Morotoge shrimp (Pandalopsis japonica): cloning and expression of eyestalk and pericardial organ isoforms produced by alternative splicing and a novel type I CHH with predicted structure shared with type II CHH peptides. Comp Biochem Physiol B 2012;162:88–9.

7

[17] Katayama H, Ohira T, Aida K, Nagasawa H. Significance of a carboxyl-terminal amide moiety in the folding and biological activity of crustacean hyperglycemic hormone. Peptides 2002;23:1537–46. [18] Katayama H, Chung JS. The specific binding sites of eyestalk- and pericardial organ-crustacean hyperglycaemic (CHHs) in multiples tissues of the blue crab, Callinectes sapidus. J Exp Biol 2009;212:542–9. [19] Kung P, Wu S, Nagaraju GPC, Tsai W, Lee C. Crustacean hyperglycemic hormone precursor transcripts in the hemocytes of the crayfish Procambarus clarkii: novel sequence characteristics relating to gene splicing pattern and transcript stability. Gen Comp Endocrinol 2013;186:80–4. [20] Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680–5. ˜ ME. Cloning and expression of hyperglycemic [21] Lago-Lestón A, Ponce E, Munoz (CHH) and molt-inhibiting (MIH) hormones mRNAs from the eyestalk of shrimps of Litopenaeus vannamei grown in different temperature and salinity conditions. Aquaculture 2007;270:343–57. [22] Lee KJ, Doran RM, Mykles DL. Crustacean hyperglycemic hormone from the tropical land crab, Gecarcinus lateralis: cloning, isoforms, and tissue expression. Gen Comp Endocrinol 2007;154:174–83. [23] Lee C, Tsai K, Tsai W, Jiang J, Chen Y. Crustacean hyperglycemic hormone: structural variants, physiological function, and cellular mechanisms of action. J Mar Sci Technol 2014;22:75–81. [24] Marco HG, Brandt W, Stoeva S, Voelter W, Gäde G. Primary structures of a second hyperglycemic peptide and of two truncated forms in the spiny lobster, Jasus lalandii. Peptides 2000;21:19–27. [25] Mettulio R, Giulianini PG, Ferrero EA, Lorenzon S, Edomi P. Functional analysis of crustacean hyperglycemic hormone by in vivo assay with wild-type and mutant recombinant proteins. Regul Peptides 2004;119:189–97. [26] Mosco A, Edomi P, Guarnaccia C, Lorenzon S, Pongor S, Ferrero EA, et al. Functional aspects of cHH C-terminal amidation in crayfish species. Regul Peptides 2008;147:88–95. [27] Nagai C, Asazuma H, Nagata S, Ohira T, Nagasawa H. A convenient method for preparation of biological active recombinant CHH of the kuruma prawn. Marsupenaeus japonicus, using the bacterial expression system. Peptides 2009;30:507–17. [28] Ohira T, Katayama H, Aida K, Nagasawa H. Expression of a recombinant crustacean hyperglycemic hormone of the kuruma prawn Penaeus japonicus in methylotrophic yeast Pichia pastoris. Fish Sci 2003;69:95–100. [29] Ohira T, Tsutsui N, Nagasawa H, Wilder MN. Preparation of two recombinant crustacean hyperglycemic hormone from the giant fresh water prawn. Macrobrachium rosenbergii, and their hyperglycemic activities. Zool Sci 2006;23:383–91. [30] Sánchez-Castrejón E, Ponce-Rivas E, Aguilar MB, Díaz F. Molecular cloning and expression of a putative crustacean hyperglycemic hormone of Litopenaeus vannamei in Pichia pastoris. Electron J Biotechnol 2008;11(4):1–9. [31] Santos EA, Maia NLE, Keller R, Gonc¸alves AA. Evidence for the involvement of the crustacean hyperglycemic hormone in the regulation of lipid metabolism. Physiol Zool 1997;70(4):415–20. [32] Schägger H. Tricine SDS-PAGE. Nat Protocols 2006;1(1):16–23. [33] Serrano L, Blanvillain G, Soyez D, Charmantier G, Grousset E, Aujoulat F, et al. Putative involvement of crustacean hyperglycemic hormone isoforms in the neuroendocrine mediation of osmoregulation in the crayfish Astacus leptodactylus. J Exp Biol 2003;206:979–88. [34] Serrano L, Grousset E, Charmantier G, Spanings-Pierrot C. Occurrence of L- and D-crustacean hyperglycemic hormone isoforms in the eyestalk x-organ/sinus gland complex during the ontogeny of the crayfish Astacus leptodactylus. J Histochem Cytochem 2004;52(9):1129–40. [35] Soyez D, Toullec J, Ollivaux C, Géraud G. l to d amino acid isomerization in a peptide hormone is a late post-translational event occurring in specialized neurosecretory cells. J Biol Chem 2000;275(48):37870–5. [36] Spanings-Pierrot C, Soyez D, Van Herp F, Gompel M, Skaret G, Grousset E, et al. Involvement of crustacean hyperglycemic hormone in the control of gill ion transport in the crab Pachygrapsus marmoratus. Gen Comp Endocrinol 2001;19:340–50. [37] Tiu SHK, He J, Chan S. The LvCHH-ITP gene of shrimp (Litopenaeus vannamei) produces a widely expressed putative ion transport peptide (LvITP) for osmoregulation. Gene 2007;396:226–35. [38] Toullec J, Serrano L, Lopez P, Soyez D, Spanings-Pierrot C. The crustacean hyperglycemic hormones from an euryhaline crab Pachigrapsus marmoratus and a fresh water crab Potamon ibericum: eyestalk and pericardial isoforms. Peptides 2006;27(6):1269–80. [39] Treerattrakool S, Udomkit A, Eurwilaichitr L, Sonthayanon B, Panyim S. Expression of biologically active crustacean hyperglycemic hormone (CHH) of Penaeus monodon in Pichia pastoris. Mar Biotechnol 2003;5:373–9. [40] Treerattrakool S, Boonchoy C, Urtagram S, Panyim S, Udomkit A. Functional characterization of recombinant gonad-inhibiting hormone (GIH) and implication of antibody neutralization on induction ovarian maturation in marine shrimp. Aquaculture 2014;428–429:166–73. [41] Tsai K, Chang S, Wu H, Shih H, Chen C, Lee C. Molecular cloning and differential expression pattern of two structural variants of the crustacean hyperglycemic hormone family from the mud crab Scylla olivacea. Gen Comp Endocrinol 2008;159:16–25. [42] Tsutsui N, Katayama H, Ohira T, Nagasawa H, Wilder MN, Aida K. The effects of crustacean hyperglycemic hormone-family peptides on vitellogenin gene expression in the kuruma prawn, Marsopenaeus japonicus. Gen Comp Endocrinol 2005;144:232–9.

Please cite this article in press as: Camacho-Jiménez L, et al. Hyperglycemic activity of the recombinant crustacean hyperglycemic hormone B1 isoform (CHH-B1) of the Pacific white shrimp Litopenaeus vannamei. Peptides (2015), http://dx.doi.org/10.1016/j.peptides.2015.05.014

553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638

G Model PEP 69488 1–8 8 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656

ARTICLE IN PRESS L. Camacho-Jiménez et al. / Peptides xxx (2015) xxx–xxx

[43] Udomkit A, Treerattrakool S, Panyim S. Crustacean hyperglycemic hormones of Penaeus monodon: cloning, production of recombinant hormones and their expression in various shrimp tissues. J Exp Mar Biol Ecol 2004;298:79–91. [44] Vargas-Albores JL, Ochoa JL. Variation of pH, osmolality, sodium and potassium concentrations in the haemolymph of sub-adult blue shrimp (Penaeus stylirostris) according to size. Comp Biochem Physiol 1992;102A(1):1–5. [45] Wanlem S, Supamattaya K, Tantikitti C, Prasertsan P, Graidist P. Expression and applications of recombinant crustacean hyperglycemic hormone from eyestalks of white shrimp (Litopenaeus vannamei) against bacterial infection. Fish Shellfish Immunol 2011;30:877–85. [46] Webster SG, Keller R, Dircksen H. The CHH-superfamily of multifunctional peptide hormones controlling crustacean metabolism, osmoregulation, moulting, and reproduction. Gen Comp Endocrinol 2012;175:217–33. [47] Wu S, Chen Y, Huang S, Tsai W, Wu H, Hsu T, et al. Demonstration of expression of a neuropeptide-encoding gene in crustacean hemocytes. Comp Biochem Physiol A 2012;161(4):463–8. [48] Wu H, Tsai W, Huang S, Chen Y, Chen Y, Hsieh Y, et al. Identification of the crustacean hyperglycemic hormone (CHH) and CHH-like peptides in the crayfish Procambarus clarkii and localization. Zool Stud 2012;51(3):288–97.

[49] Xie X, Zhu D, Yang J, Qiu X, Cui X, Tang J. Molecular cloning of two structure variants of crustacean hyperglycemic hormone (CHH) from the swimming crab (Portunus trituberculatus), and their gene expression during molting and ovarian development. Zool Sci 2014;31(12):802–9. [50] Yasuda A, Yasuda Y, Fujita T, Naya Y. Characterization of crustacean hyperglycemic hormone from crayfish (Procambarus clarkii): multiplicity of molecular forms by stereoinversion and diverse functions. Gen Comp Endocrinol 1994;95:387–98. [51] Yodmuang S, Udomkit A, Treerattrakool S, Panyim S. Molecular and biological characterization of molt-inhibiting hormone of Penaeus monodon. J Exp Mar Biol Ecol 2004;312:101–14. [52] Zarubin TP, Chang ES, Mykles DL. Expression of recombinant eyestalk crustacean hyperglycemic hormone from the tropical land crab. Gecarcinus lateralis, that inhibits Y-organ edysteroidogenesis in vitro. Mol Biol Rep 2009;36:1231–7. [53] Zheng J, Chen HY, Choi CY, Roer RD, Watson RD. Molecular cloning of a putative crustacean hyperglycemic hormone (CHH) isoform from extra-eyestalk tissue of the blue crab (Callinectes sapidus), and determination of temporal and spatial patterns of CHH gene expression. Gen Comp Endocrinol 2010;169:174–81.

Please cite this article in press as: Camacho-Jiménez L, et al. Hyperglycemic activity of the recombinant crustacean hyperglycemic hormone B1 isoform (CHH-B1) of the Pacific white shrimp Litopenaeus vannamei. Peptides (2015), http://dx.doi.org/10.1016/j.peptides.2015.05.014

657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675