339
BlOchmllca et BlOph.plCll Acta. 1077 (1991) 339-345 '-' 1991 Elsevier Science Puhllshers B.V. 0167-4838/91/$03.50 ADONIS 0167483891001691
BBAPRO 33889
Determination of the primary and secondary structures of the dromedary ( Came/us dromedarius) prolactin and comparison with prolactins from other species Nadine Martinat J
I,
Jean-Claude Huet 2, Claude Nespoulous and Jean-Claude Pemallet 2
2,
Yves Cambamaus
1
Vmte Endocrmofog,e de fa Gametogenese. INRA & URA eN RS 1291. StatIOn de PhyslOlogle de fa ReproductIOn. Nouzll(v (France) and .' LahoratOlre dOEtude des Protemes, DeparJemenl de PhY'iw!ogle et BlOchlmle veKetales. JN RA - Versailles. Vf?rsmllf?s (Frana')
(Received 28 August 1991)
Key words: Prolactin; Amino acid
sequence~
Glycosylation site: Secondary structure: (Dromedary)
A non-glycos)lated form of camel prolactin (camPRL), isolated from one-humped camel (Came/us dromedariu.v) pituitaries, was totally sequenced. A glycosylated form, separated by affinity chromatography on ConA-Scpharose, was partially sequenced. TIle comparison of the N-terminal amino acid sequences of the glycosylated and non-glycosylated forms showed that the only putative site of N-glycosylation (Asn-3]) was indeed glycosylated. TIle far ultraviolet (UV) circular dichroism (CD) spectra of the two isohormones were identical, suggesting that the carbohydrate moiety had no effect on the global camPRL secondary structure. The far UV circular dichroism spectra of the two isohormones were analyzed in order to determine their relative proportions of periodic secondary structure, 60% of which was found to be in a-helix, as in prolactins of other species. The dromedary sequence was compared to those of other species and interpreted in term of evolutionary process. As already found for gonadotropins, the closest species to the dromedary was found to be the pig.
Introdnction Prolactin [1] together with growth hormone (GH) [2-3], placental lactogen (PL) [4], placental growth hormone [5] and other newly discovered molecules [6-9] are considered to belong to the same hormonal family as they share a number of common biological, immunological and structural features. Although most GHs, PRLs and PLs from different species are specific, some of them exhibit both lactogenic and somatotrophic activities [1]. The understanding of the structural origin of the hormonal specificity in this family of molecules requires the study of the physico-chemical and biological properties of hormones from different species. In
Abhreviations: camPRL. camel prolactin: FSH, follitropin; GH. growth hormone; hCG. human chononic gonadotropin: llI, lutropm; PL placental lactogen: PRl, prolactin: Pth-Xaao phenyl thiohy. danroin amino acid: UV. ultraviolet; CD. circular dichroism. Correspondence: J.-C. Pernallet, Laboratoire d'Etude des Proteines, 'I'RA-Ver>a,lIes. Route de 51. Cyr F-78026 Versailles CEDEX, France.
this respect. we purified camPRL [10]. the one-humped camel (Came/us dromedarius) prolactin. In this report. we present the complete sequence of a non-glycosylated form of prolactin from the dromedary. We also determined the only N-glycosylation site of a glycosylated form of camPRL and demonstrated it was indeed glycosylated. In addition, we have compared the secondary structure of these proteins using far UV circular dichroism, performed on both glycosylated and non-glycosylated camPRL. Materials and Methods Pro/actin purification CamPRL was purified as previously reported [10). Brieny, pituitary glands from one-humped camels (Came/us dromedarius) were collected in slaughter houses in southern Morocco; after extraction of lutrapin (LH) and follitropin (FSH) according to [11], the resulting residue was ground in water and lyophilized. CamPRL was extracted and separated from camGH by ion-exchange chromatography on DEAE-Trisacryl, then isoforms of different isoelectric points were isolated on
340 DEAE-Sephacel as described in [10]. An aliquot of each highly purified camPRL preparation was subjected to affinity chromatography on ConA-Sepharose in order to separate glycosylated and non-glycosylated forms. The most abundant unglycosylated (camPRL-A) and glycosylated isoforms (camPRL-CII) were used in this study. They were both extensively dialyzed and lyophilized prior further experiments. Reduction and alkylation of prolactin Proteins were reduced with 2-mercaptoethanol and alkylated with 4-vinylpyridine according to [12] before submission to enzymatic or chemical cleavage.
Lys-C and tryptic digests Using either TPCK trypsin (trypsin, EC 3.4.21.4, Sigma Ref. T8642, batch number 38F8140, treated with L-l-(tosylamido)-2-phenylethylchloromethylketone) or Lys-C endoproteinase (EC 3.4.99.30, Boehringer Ref. 476986), 0.2 mg of reduced and alkylated prolactin, dissolved (1 mg ml-') in 0.2 M ammonium hydrogenocarbonate buffer (pH 8.0) were cleaved after incubation for 18 h at 22 C (enzyme to substrate weight ratio 1/50). The digestion was stopped by acidification to pH 2 to 3 with 10% trifluoroacetic acid. Q
Asp-N digests Asp-N endoproteinase, which allows the cleavage at the amino side of Asp and CysSO,H [13], purchased from Boehringer (Ref. 1054589, batch number 11201320-01), was used for the cleavage of 0.2 mg of prolactin (1 mg ml-') with an enzyme to substrate weight ratio of 1/100, for 18 h at 37°C in 50 mM sodium phosphate buffer (pH 8). lodosobenzoic acid digests Iodosobenzoic peptides were obtained according to [14] with 0.5 mg of reduced and alkylated protein.
Isolation of digested peptides The fractionation of peptides by reversed-phase HPLC was conducted with a CU 300 Octyl Aquapore 30 X 4.6 mm column at room temperature on a SP 8800 Spectra Physics equipment with a UV diode array detector. Solvent A was MilliporeT\1-treated water with 0.1% CF3COOH and solvent B 40% MilIipore™-treated water, 60% CH 3CN (FISONS, far UV grade) and 0.09% CF3COOH. The following gradient was used at a flowrate of 0.5 m] min - L from 100% of solvent A to 100% of solvent B in 60 min, 5 min at this constant concentration, from 100% B to 100% A in 10 min and 10 min of re-equilibration at 100% of solvent A.
Amilia acid ana/vsls Native glyco~ylated and unglycosylated prolactins (1 nmol) were submitted to hydrochloric acid hydrolysis in vapour phase at 115°C for 24 h before amino acid analysis using a Biotronik LC 5001 device with lithium buffers. Sulphur amino acids were determined after performic oxidation according to Henschen [13]. The tryptophan content was determined using the spectrophotometric method of Bencze and Schmid [15]. Protein sequencllll( Automated Edman degradation of the whole protein and of peptides was performed following the original method of Hewick et al. [161 using an Applied Biosysterns 475A sequcncer and its on line phenyl thiohydantoin amino acid (Pth-Xaa) analyzer model 120A with reagents and methods of the manufacturer. Homology search in data bank Search for homologies between camPRL and other proteins was pcrformed using the method of Kanehisa [17] with the following libraries: National Biomedical Research Foundation (release 23) and the translation of the open reading frames of Genbank (release 62) and European Molecular Biology Library (release 21 + ) available at the French data base BISANCE. All computations were done using the similarity matrix of Dayhoff et al. [18]. Circlliar dichroism CD spectra were recorded at 1 nm interval over the wavelength range 180-260nm, using a Mark V JobinYvon dichrograph, calibrated using iso-androsterone (Roussel-Uelaf). The optical rotation was checked with cytochrome C (horse heart). lysozyme (chicken egg white) and L-Iactate dehydrogenase (rabbit muscle). CD measurements were made at room temperature. Glycosylated and unglycosylated prolactin solutions (0.6 mg I ml- ) were made in 50 mM Tris-HCl (pH 9.3). Protein samples were placed in a 0.01 cm pathlength cell. Five reproducible spectra were collected and averaged for each sample. The nct spectrum of each protein was obtained by subtracting base line obtained with pure water. The CD data were ex.pressed as the mean residue ellipcity and the spectra analyzed following the method of Yang et al. [19] for the determination of the contribution of the different secondary structures. This method is based on the least square fitting of the measured spectrum, adjusted to the weighted sum of pure spectra corresponding to the different secondary structures, spectra deduced from known three-dimensional structures of proteins. The method was tested with the same standard proteins as mentioned above. The results were indicated with their confidence limits (P ~ 0.95) obtained after curve fitting computation.
341 Results
1~
2~
glytolylallon
lit!!
3~t
1~
5~
LP I CPSGAUNCQUSLADLFDRRU I LSHV I HNLSSEMFHEFDKAVAOGAGF
Isolation and sequencing of prolaclin peptide fragments After chemical or enzyme digestions, several peptides were obtained from unglycosylated prolactin. Thcir re. versed·phase HPLC chromatograms (peptide maps) are
-;-----KCS· -------;- N.t.r---~'::~ANI. 6~ ----~
~·--T-·-·-.
KC6-
B
:a
CH3CN% W2
~
m
"
tijO.05
50
of
. ~
~
on
c
CH3CN%
o
CH3CN%
NO.1
;; !l
.a
I ~
on
Oi ;; m
I!
.e ~
0.1
Fig. 1. Reversed-phase HPLC chromatograms of peptides resulting from the prolactin cleavage (peptide maps). Peptides are identified u
BlOchmllca et BlOph.plCll Acta. 1077 (1991) 339-345 '-' 1991 Elsevier Science Puhllshers B.V. 0167-4838/91/$03.50 ADONIS 0167483891001691
BBAPRO 33889
Determination of the primary and secondary structures of the dromedary ( Came/us dromedarius) prolactin and comparison with prolactins from other species Nadine Martinat J
I,
Jean-Claude Huet 2, Claude Nespoulous and Jean-Claude Pemallet 2
2,
Yves Cambamaus
1
Vmte Endocrmofog,e de fa Gametogenese. INRA & URA eN RS 1291. StatIOn de PhyslOlogle de fa ReproductIOn. Nouzll(v (France) and .' LahoratOlre dOEtude des Protemes, DeparJemenl de PhY'iw!ogle et BlOchlmle veKetales. JN RA - Versailles. Vf?rsmllf?s (Frana')
(Received 28 August 1991)
Key words: Prolactin; Amino acid
sequence~
Glycosylation site: Secondary structure: (Dromedary)
A non-glycos)lated form of camel prolactin (camPRL), isolated from one-humped camel (Came/us dromedariu.v) pituitaries, was totally sequenced. A glycosylated form, separated by affinity chromatography on ConA-Scpharose, was partially sequenced. TIle comparison of the N-terminal amino acid sequences of the glycosylated and non-glycosylated forms showed that the only putative site of N-glycosylation (Asn-3]) was indeed glycosylated. TIle far ultraviolet (UV) circular dichroism (CD) spectra of the two isohormones were identical, suggesting that the carbohydrate moiety had no effect on the global camPRL secondary structure. The far UV circular dichroism spectra of the two isohormones were analyzed in order to determine their relative proportions of periodic secondary structure, 60% of which was found to be in a-helix, as in prolactins of other species. The dromedary sequence was compared to those of other species and interpreted in term of evolutionary process. As already found for gonadotropins, the closest species to the dromedary was found to be the pig.
Introdnction Prolactin [1] together with growth hormone (GH) [2-3], placental lactogen (PL) [4], placental growth hormone [5] and other newly discovered molecules [6-9] are considered to belong to the same hormonal family as they share a number of common biological, immunological and structural features. Although most GHs, PRLs and PLs from different species are specific, some of them exhibit both lactogenic and somatotrophic activities [1]. The understanding of the structural origin of the hormonal specificity in this family of molecules requires the study of the physico-chemical and biological properties of hormones from different species. In
Abhreviations: camPRL. camel prolactin: FSH, follitropin; GH. growth hormone; hCG. human chononic gonadotropin: llI, lutropm; PL placental lactogen: PRl, prolactin: Pth-Xaao phenyl thiohy. danroin amino acid: UV. ultraviolet; CD. circular dichroism. Correspondence: J.-C. Pernallet, Laboratoire d'Etude des Proteines, 'I'RA-Ver>a,lIes. Route de 51. Cyr F-78026 Versailles CEDEX, France.
this respect. we purified camPRL [10]. the one-humped camel (Came/us dromedarius) prolactin. In this report. we present the complete sequence of a non-glycosylated form of prolactin from the dromedary. We also determined the only N-glycosylation site of a glycosylated form of camPRL and demonstrated it was indeed glycosylated. In addition, we have compared the secondary structure of these proteins using far UV circular dichroism, performed on both glycosylated and non-glycosylated camPRL. Materials and Methods Pro/actin purification CamPRL was purified as previously reported [10). Brieny, pituitary glands from one-humped camels (Came/us dromedarius) were collected in slaughter houses in southern Morocco; after extraction of lutrapin (LH) and follitropin (FSH) according to [11], the resulting residue was ground in water and lyophilized. CamPRL was extracted and separated from camGH by ion-exchange chromatography on DEAE-Trisacryl, then isoforms of different isoelectric points were isolated on
340 DEAE-Sephacel as described in [10]. An aliquot of each highly purified camPRL preparation was subjected to affinity chromatography on ConA-Sepharose in order to separate glycosylated and non-glycosylated forms. The most abundant unglycosylated (camPRL-A) and glycosylated isoforms (camPRL-CII) were used in this study. They were both extensively dialyzed and lyophilized prior further experiments. Reduction and alkylation of prolactin Proteins were reduced with 2-mercaptoethanol and alkylated with 4-vinylpyridine according to [12] before submission to enzymatic or chemical cleavage.
Lys-C and tryptic digests Using either TPCK trypsin (trypsin, EC 3.4.21.4, Sigma Ref. T8642, batch number 38F8140, treated with L-l-(tosylamido)-2-phenylethylchloromethylketone) or Lys-C endoproteinase (EC 3.4.99.30, Boehringer Ref. 476986), 0.2 mg of reduced and alkylated prolactin, dissolved (1 mg ml-') in 0.2 M ammonium hydrogenocarbonate buffer (pH 8.0) were cleaved after incubation for 18 h at 22 C (enzyme to substrate weight ratio 1/50). The digestion was stopped by acidification to pH 2 to 3 with 10% trifluoroacetic acid. Q
Asp-N digests Asp-N endoproteinase, which allows the cleavage at the amino side of Asp and CysSO,H [13], purchased from Boehringer (Ref. 1054589, batch number 11201320-01), was used for the cleavage of 0.2 mg of prolactin (1 mg ml-') with an enzyme to substrate weight ratio of 1/100, for 18 h at 37°C in 50 mM sodium phosphate buffer (pH 8). lodosobenzoic acid digests Iodosobenzoic peptides were obtained according to [14] with 0.5 mg of reduced and alkylated protein.
Isolation of digested peptides The fractionation of peptides by reversed-phase HPLC was conducted with a CU 300 Octyl Aquapore 30 X 4.6 mm column at room temperature on a SP 8800 Spectra Physics equipment with a UV diode array detector. Solvent A was MilliporeT\1-treated water with 0.1% CF3COOH and solvent B 40% MilIipore™-treated water, 60% CH 3CN (FISONS, far UV grade) and 0.09% CF3COOH. The following gradient was used at a flowrate of 0.5 m] min - L from 100% of solvent A to 100% of solvent B in 60 min, 5 min at this constant concentration, from 100% B to 100% A in 10 min and 10 min of re-equilibration at 100% of solvent A.
Amilia acid ana/vsls Native glyco~ylated and unglycosylated prolactins (1 nmol) were submitted to hydrochloric acid hydrolysis in vapour phase at 115°C for 24 h before amino acid analysis using a Biotronik LC 5001 device with lithium buffers. Sulphur amino acids were determined after performic oxidation according to Henschen [13]. The tryptophan content was determined using the spectrophotometric method of Bencze and Schmid [15]. Protein sequencllll( Automated Edman degradation of the whole protein and of peptides was performed following the original method of Hewick et al. [161 using an Applied Biosysterns 475A sequcncer and its on line phenyl thiohydantoin amino acid (Pth-Xaa) analyzer model 120A with reagents and methods of the manufacturer. Homology search in data bank Search for homologies between camPRL and other proteins was pcrformed using the method of Kanehisa [17] with the following libraries: National Biomedical Research Foundation (release 23) and the translation of the open reading frames of Genbank (release 62) and European Molecular Biology Library (release 21 + ) available at the French data base BISANCE. All computations were done using the similarity matrix of Dayhoff et al. [18]. Circlliar dichroism CD spectra were recorded at 1 nm interval over the wavelength range 180-260nm, using a Mark V JobinYvon dichrograph, calibrated using iso-androsterone (Roussel-Uelaf). The optical rotation was checked with cytochrome C (horse heart). lysozyme (chicken egg white) and L-Iactate dehydrogenase (rabbit muscle). CD measurements were made at room temperature. Glycosylated and unglycosylated prolactin solutions (0.6 mg I ml- ) were made in 50 mM Tris-HCl (pH 9.3). Protein samples were placed in a 0.01 cm pathlength cell. Five reproducible spectra were collected and averaged for each sample. The nct spectrum of each protein was obtained by subtracting base line obtained with pure water. The CD data were ex.pressed as the mean residue ellipcity and the spectra analyzed following the method of Yang et al. [19] for the determination of the contribution of the different secondary structures. This method is based on the least square fitting of the measured spectrum, adjusted to the weighted sum of pure spectra corresponding to the different secondary structures, spectra deduced from known three-dimensional structures of proteins. The method was tested with the same standard proteins as mentioned above. The results were indicated with their confidence limits (P ~ 0.95) obtained after curve fitting computation.
341 Results
1~
2~
glytolylallon
lit!!
3~t
1~
5~
LP I CPSGAUNCQUSLADLFDRRU I LSHV I HNLSSEMFHEFDKAVAOGAGF
Isolation and sequencing of prolaclin peptide fragments After chemical or enzyme digestions, several peptides were obtained from unglycosylated prolactin. Thcir re. versed·phase HPLC chromatograms (peptide maps) are
-;-----KCS· -------;- N.t.r---~'::~ANI. 6~ ----~
~·--T-·-·-.
KC6-
B
:a
CH3CN% W2
~
m
"
tijO.05
50
of
. ~
~
on
c
CH3CN%
o
CH3CN%
NO.1
;; !l
.a
I ~
on
Oi ;; m
I!
.e ~
0.1
Fig. 1. Reversed-phase HPLC chromatograms of peptides resulting from the prolactin cleavage (peptide maps). Peptides are identified u
