The molecular characterisation of chicken pituitary N-terminal pro-opiomelanocortin (POMC)

Molecular and Cellular Endocrinology 142 (1998) 119 – 130

The molecular characterisation of chicken pituitary N-terminal pro-opiomelanocortin (POMC) L.R. Berghman a,*, B. Devreese c, P. Verhaert b, H. Gerets a, L. Arckens a, J. Vanden Broeck b, J. Van Beeumen c, H. Vaudry d, F. Vandesande a a

Laboratory of Neuroendocrinology and Immunological Biotechnology, Zoological Institute, Naamsestraat 59, B-3000 Leu6en, Belgium b Laboratory of De6elopmental Physiology and Molecular Biology, Zoological Institute, Naamsestraat 59, B-3000 Leu6en, Belgium c Laboratory of Protein Biochemistry and Protein Engineering, Uni6ersity of Ghent, Ghent, Belgium d Laboratory of Cellular and Molecular Neuroendocrinology, Inserm U413, IFRMP n° 23, Uni6ersity of Rouen, Rouen, France Received 2 March 1998; accepted 11 May 1998

Abstract Monoclonal antibodies (Mabs) specifically recognizing the chicken pituitary corticotropes were used to isolate a population of closely related peptides from crude chicken pituitary extracts. A homogeneous N-terminal sequence homologous to the extreme N-terminus of mammalian and amphibian pro-opiomelanocortin (POMC) was revealed. Further physicochemical analysis proved the existence of a series of C-terminally truncated peptides including 3 major molecular species corresponding to Ser1 –Gly64, Ser1–Arg73 and Ser1 –Gly105 respectively. The two latter molecules were shown to be N-glycosylated at position Asn67, with mass spectrometric data indicating a carbohydrate structure of the oligomannose 5 type, in addition to two more complex structures. No evidence was found in favour of O-glycosylation on Ser47. Degenerated PCR primers were deduced from the above protein sequence and from the known chicken adrenocorticotropic hormone (ACTH) sequence. The nucleotide sequence obtained by reversed transcription PCR (RT-PCR) completely confirmed the new amino acid sequence data including pro-g-MSH, the joining peptide and ACTH. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Chicken pituitary; Pro-opiomelanocortin; N-terminal; Isoforms; Sequence

1. Introduction Pro-opiomelanocortin (POMC) is the multifunctional precursor protein of a number of hormonally active peptides that are secreted upon excision by specific endoproteases. POMC derivatives produced in the anterior pituitary gland include the N-terminal fragment, a joining peptide (JP), adrenocorticotropin (ACTH) and b-lipotropin (b-LPH). ACTH can be further cleaved into a-MSH and corticotropin-like intermediate lobe peptide (CLIP) in the pars intermedia * Corresponding author. Present address: Department of Poultry Science, 418 Kleberg center, Texas A&M University, College Station, Texas 77843-2472, USA; tel.: 32 16 323951; fax: 32 16 323902; e-mail: [email protected]

of the pituitary, whereas b-LPH can be further cleaved into g-LPH and b-endorphin (END). The N-terminal peptide is also being referred to as pro-g-MSH, which has in the rat been shown to be co-secreted with ACTH from the pituitary in an inactive form. Peptides originating from the N-terminal POMC peptide include a mitogenic factor (POMC 1–48/49) and g3-MSH (POMC 50–74). As compared to other vertebrate classes, relatively little has been reported about the biochemistry of avian POMC. Although protein purifications have been reported for a number of chicken POMC-derived hormones, no nucleotide sequence information is available to date. Purified avian POMC derivatives include an N-terminal peptide in ostrich (Naude´ et al., 1993), ACTH in chicken (Hayashi and Imai, 1991), in turkey

0303-7207/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0303-7207(98)00112-9

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(Chang et al., 1980) and in ostrich (Naude´ and Oelofsen, 1977; Li et al., 1978) and b-LPH in ostrich (Naude´ et al., 1981) and in turkey (Chang et al., 1980). In this study, we report the purification and characterization of a new family of N-terminal chicken POMC peptides. The obtained protein sequence data were further used to determine the complete nucleotide sequence of N-terminal POMC, the joining peptide and ACTH in chicken by RT-PCR.

2. Materials and methods

2.1. Monoclonal antibody production Monoclonal antibodies against an affinity-purified mixture of chicken pituitary glycoproteins were produced as described before (Berghman et al., 1988; Vandesande and Berghman, 1988). Briefly, mice were immunized with the glycoprotein mixture and activated splenocytes were fused with SP2/0 myeloma cells by electrofusion (De Boer et al., 1989). Hybridomas were screened for antibody production by a miniaturized immunocytochemical protocol on sagittal paraffin sections of Bouin–Hollande sublimate-fixed chicken pituitaries (Berghman et al., 1989). Immunopositive cultures, producing the desired immunocytochemical staining picture (Section 2.2) were cloned by limiting dilution and used for ascites production. The hybridoma line that produced the present results was designated 16D9.

2.2. Immunocytochemistry Antibody production was routinely monitored by immunocytochemistry on 7 mm paraffin sections of control pituitaries that had been fixed by immersion in Bouin–Hollande sublimate immediately after dissection. Embedding, sectioning and dewaxing were performed according to routine laboratory protocols. Primary antibodies were left on the sections either for 5 h (in a 1-day staining protocol) or overnight. Primary antibodies were detected by a 1 h incubation with the appropriate peroxidase-conjugated secondary antibodies (Dakopatts, Glostrup, Denmark). DAB and peroxide were used as substrate system. For comparison, transverse paraffin sections of rat pituitary were stained with the same protocol.

2.3. Immunoaffinity chromatography Monoclonal antibodies were purified by protein A affinity chromatography, dialyzed and coupled to CNBr-activated Sepharose 4B (Pharmacia, Uppsala, Sweden). Pituitaries were collected in a slaughterhouse and snap frozen in liquid nitrogen. The tissue was

homogenized in ice-cold 50 mM Tris–HCl buffer, pH 9.0 (20 ml buffer per g tissue) in the presence of a wide spectrum of enzyme inhibitors, i.e. one tablet ‘Complete’ (Boehringer, Mannheim, Germany) per 50 ml of homogenate, added immediately after homogenization. The extract was stirred for 4 h at 4°C, centrifuged and its pH adjusted to 7.0 with diluted HCl. The equivalent of 100 pituitaries was applied to a 2-ml immunoadsorbent and recycled for 1 h. The column was then rinsed to baseline absorption at 280 nm with 50 mM Tris– HCl buffer, pH 7.6, containing 150 mM NaCl and finally eluted with a glycine–HCl buffer, pH 2.8, also containing 150 mM NaCl. The eluent was immediately neutralized by addition of 3 M Tris–HCl, pH 8, and frozen for further analysis.

2.4. RP-HPLC and amino acid sequence determination The immunoaffinity-purified material was desalted and tentatively fractionated on a 4.6× 100 mm polystyrene/divinylbenzene polymer column (POROS) by use of a linear gradient from 0.1% (v/v) trifluoroacetic acid (TFA) in MilliQ water to 0.12% TFA in 60% acetonitrile over 30 min at a flow rate of 2 ml/min. Solvents were delivered with a Waters 6000 E system controller and detection was performed at 210 nm. Four fractions were collected of which the major peak in the chromatogram (designated ‘RP-4’) was dried in a Savant vacuum centrifuge and used for automatic Edman degradation. In order to get internal sequence information, the same material was carboxymethylated and enzymatically digested by use of Lys-C endoprotease (Boehringer) in 50 mM Tris–HCl at pH 8.5 and Glu-C-endoprotease from Staphylococcus aureus (Miles, Stokes, UK) in 0.1 M NH4HCO3 at pH 7.5 respectively. The digests were then purified by RP-HPLC on a 2.1× 250 mm Altima C18 column (Alltech, Deerfield, IL) using a gradient from 0.1% TFA in 5% acetonitrile to 0.1% TFA in 70% acetonitrile in 65 min at a flow rate of 200 ml/min. The resulting peaks were vacuum dried and submitted to Edman degradation and mass spectrometry. Peptide sequencing was performed on either a 477A or a 476A pulsed liquid protein sequencer (both Perkin Elmer, Applied Biosystems Division, Foster City, CA). Fractions of the samples were submitted to electrospray mass spectrometry using a VG Bio-Q mass spectrometer (Micromass, Manchester, UK). Therefore, the samples were diluted in 50% acetonitrile/1% formic acid in water and injected into the source of the mass spectrometer using a flow rate of 6 ml/min delivered by a Hamilton Syringe Pump 11 (Harvard InstK, South Natick, MA). The sample cone voltage was set at 40 V. Nine second scans ranging from 500 to 1500 Da were accumulated during 2.5 min. The spectra were collected

L.R. Berghman et al. / Molecular and Cellular Endocrinology 142 (1998) 119–130

and processed using software delivered with the instrument.

2.5. SDS-PAGE Electrophoretic analysis of the 4 RP-fractions of the immunoaffinity-purified mixture was performed by Tricine SDS-PAGE on 160×160 ×1.5 mm slab gels (16.5%T, 3%C) as described by Scha¨gger and von Jagow (1987). These were either stained by Coomassie Brilliant Blue 250-G (0.025% in 10% acetic acid) or blotted onto a PVDF membrane (Bio-Rad, Hercules, CA) by semi-dry electroblotting. The blotted proteins were visualized by staining with 0.1% Coomassie Blue 250-R in 50% methanol and destaining in 50% methanol/10% acetic acid. The bands were individually excised from the membrane for N-terminal amino-acid determination in an automated gas phase sequenator (LF 3600 TC Protein Sequencer, Beckman, Fullerton, CA). Alternatively, the major RP-HPLC peak (designated ‘‘RP-4’’) was further separated on a 60× 0.75 cm TSKG2000SW high pressure gel permeation column (Pharmacia) with PBS as the mobile phase (0.5 ml/min). The purification process was monitored by discontinuous SDS-PAGE for small MW peptides according to Scha¨gger and von Jagow (1987).

2.6. Enzymatic deglycosylation Twenty micrograms of the lyophilized RP-fractionated immunoaffinity-purified material (peak 4) was digested by glycopeptidase F from Fla6obacterium meningosepticum (PNGase F, EC 3.2.2.18, Boehringer) according to Tarentino et al. (1985), in order to specifically remove potential N-linked carbohydrate moieties from the peptide backbone. Briefly, the peptide mixture was first denatured and reduced by boiling in 0.5% SDS 0.1 M b-mercaptoethanol. PNGase F was then added to a final concentration of 10 U/ml in a 50 mM Tris –phosphate buffer, pH 8.6, containing 10 mM 1,10-phenantroline, 33 mM b-mercaptoethanol, 0.17% SDS and 1.25% Nonidet P-40. After 24 h of digestion at 37°C, the material was further diluted with electrophoresis sample buffer and separated electrophoretically according to Scha¨gger and von Jagow (1987). Apparent molecular weights were calculated using the software provided by the Image Master (Pharmacia).

2.7. RT-PCR In order to verify the obtained amino acid sequence, the corresponding N-terminal region of the mRNA was amplified by RT-PCR. Poly-A RNA was prepared from 100 mg of frozen pituitaries by use of the ‘Quick-

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Prep Micro mRNA Purification kit’ (Pharmacia). RTPCR was performed by employing the ‘GeneAmp RNA PCR kit’ (Perkin Elmer, Norwalk, CT). The oligo-dT and random hexamer primers supplied with the kit were used for reverse transcription of the mRNA into cDNA. The sequences of the upstream and downstream PCR primers were based on known POMC-derived peptide sequences (N-terminal part and ACTH, respectively). RT-PCR was performed according to the instructions of the kit. The PCR-cycle consisted of a denaturation step (95°C, 45 s), an annealing step (55°C, 60 s) and an elongation step (72°C, 60 s). After 35 cycles, the final elongation step was prolonged for 7 min. PCR-fragments were analyzed by horizontal agarose electrophoresis and visualized via ethidiumbromide fluorescence. The PCR-primers (supplied by GibcoBRL Life Technologies, Merelbeke, Belgium) were designed as follows: “ 5%GCAGATCTAGA (T,A)(G,C)I GGI CCI TG(T,C) TGG GA(G,A) AA 3%; ’ 5%GCGAGCTCGAG C(G,T) I(C,T)(G,T) (A,G)AA (T,C)TC CAT IGG (A,G)TA 3%.

2.8. Cloning and sequencing The PCR-fragment was ligated into a TA-cloning vector (pCR™ 2.1, ‘Original TA-Cloning kit’, InvitroGen, Carlsbad, CA). INVaF% cells were transformed with the resulting ligation mixture and recombinant bacterial colonies were selected by blue/white screening. Recombinant plasmid DNA was purified with the ‘High Pure Plasmid Isolation kit’ from Boehringer and sequenced on both strands by using the ‘Sequenase version 2.0 DNA sequencing kit’ (Amersham-USB, Buckinghamshire, UK) which is based on the dideoxy chain termination method (Sanger et al., 1977).

3. Results

3.1. Immunocytochemical localization The obtained monoclonal antibodies (Mabs) were tentatively subdivided into different specificity categories on the basis of their immunocytochemical characteristics. As described earlier, some Mabs recognized the somatotropes in the caudal lobe (Berghman et al., 1987) and others stained the gonadotropic either alone or along with the thyrotropic cells (Berghman et al., 1993). Finally, some Mabs stained a typical cell population with a patch-like distribution throughout the cephalic lobe only (Fig. 1(A)). Double staining experiments with an anti-hACTH antiserum identified these cells as the adrenocorticotropes (results not shown). The Mab which had been designated 16D9 was selected

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Fig. 1. (A) Immunocytochemical demonstration of the chicken pituitary corticotrophs using the monoclonal antibody 16D9. Corticotrophs are confined to the cephalic lobe of the adenohypophysis where they are present in a typical globule-like arrangement. CE: cephalic lobe; CA: caudal lobe. (B) Immunocytochemical staining of the rat pars intermedia by MAb 16D9. In contrast to the chicken pituitary, the corticotrophs in the pars distalis are negative. PD: pars distalis; PI: pars intermedia; PN: pars nervosa.

for further use. On transverse sections of the rat pituitary, this Mab produced a strong staining of the pars intermedia, with negligible staining in the pars distalis (Fig. 1(B)).

3.2. In6entory of isoforms. The yield of the immunoaffinity protocol was estimated at around 200 mg per 100 pituitary equivalents ( 1 g wet weight). The immunoaffinity-purified preparation was first desalted and tentatively fractionated on a POROS reversed phase (RP) column (Perseptive Biosystems, Framingham, MA). A typical RP-HPLC chromatogram (Fig. 2) shows a spectrum of closely related compounds with elution times ranging from 21 to 27 min, corresponding with 42 to 54% acetonitrile. The chromatogram was subdivided into four major fractions and lyophilized for further analysis. SDS-PAGE of these 4 RP-HPLC preparations confirmed the existence of a series of at least seven molecular species with apparent molecular masses (MM) ranging from 7 to 17 kDa (Fig. 3). The three major components are shown at apparent MM of 7 kDa (band ‘b; in peak 5, lane 4), 11kDa (band ‘d’ apparent in peaks 3 and 4, lanes 2 and 3) and 15 kDa (band ‘f’ in peak 4, lane 3). The corresponding figures obtained by mass spectrometry on the same preparations are shown in Table 1. Again, three major molecular species were detected, but this time their MM were 7, 9.5 and 13 kDa respectively, indicating an overestimation by SDS-PAGE of the MM of the two larger components by about 1.5–2 kDa.

Each of the components (both major and minor bands) was excised from an electroblot on PVDF membrane and submitted to N-terminal sequencing. Results for the individual components, as shown in Table 2, demonstrate a perfect overlap of their N-termini and considerable homology with the N-terminal portion of amphibian, mammalian and ostrich POMC. This was not true, however, in the case of a band (‘h’) with a high but unknown MM which was retained at a position far above the highest standard protein. HPLC gel permeation chromatography of peak RP-4 (Fig. 4) yielded highly purified preparations of the 15 and 11 kDa species respectively, as shown by SDSPAGE in Fig. 5. Rechromatography by RP-HPLC of the major GPC peaks confirmed their identical retention times in RP, despite the considerable difference in MM (results not shown).

3.3. Amino acid and nucleotide sequence of N-terminal POMC An overview of the amino acid sequence of N-terminal POMC from Ser1-Glu94 is depicted in Table 3. This sequence was reconstructed from the partial sequences of the different peptides that were recovered upon digestion of the carboxymethylated immunoaffinity-purified chicken NT-POMC with Lys-C endoprotease and Glu-C-endoprotease, respectively. The peptides that contributed to this reconstruction are underlined in Table 3 and are listed in Table 4. Amino acids that had not been directly sequenced were deduced from the nucleotide sequence. This also allowed

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Fig. 2. Chromatogram of the tentative fractionation of the immunoaffinity-purified peptide mixture on a POROS RP-HPLC column. Four fractions (designated RP-3 to RP-6) were collected and vacuum dried and used for further characterization.

to fill the gap between Glu95 and Ser108, which is the first amino acid of chicken ACTH (Ser108-Phe146). The nucleotide data completely confirmed the available protein sequence (Table 3).

3.4. N-deglycosylation Reversed phase peak 4 (containing basically the 11 and 15 kDa isoforms) was submitted to enzymatic digestion with PNG-ase F and the MM shifts were estimated by comparison of pre- and post-digestion compounds on reducing SDS-PAGE (Fig. 6). This re-

vealed that both the 11 and 15 kDa isoforms were N-glycosylated: their MM were reduced from 10363 Da to 8259 Da and from 14391 to 11677 kDa respectively, suggesting a comparable degree of glycosylation, with an apparent carbohydrate size corresponding to 2.1 and 2.7 kDa respectively. Mass spectrometry in combination with the obtained sequence data indicated Asn67 as the likely site for N-glycosylation. Indeed, peptide Sa7 (His57–Glu76; Table 3) which was produced by aspecific cleavage after Ser56, has a theoretical MM of 2462.7 Da, whereas experimental MM of 3676.9, 4220.1 and 4382.0 Da were measured by mass spectrometry.

3.5. Nucleotide sequence The nucleotide sequence of the RT-PCR fragment corresponds to the coding region containing the N-terminal POMC-peptide, the joining peptide and ACTH (Table 3). Its deduced amino acid sequence shows a high degree of sequence similarity with POMCs from other vertebrate species (Table 5): 87% with ostrich, 69% identity with Xenopus, 64% with Rana, 62% with Table 1 Mass spectrometric analysis of the 4 RP-HPLC fractions

Fig. 3. Electrophoretic separation of the immunoaffinity-purified peptide mixture upon fraction by RP-HPLC. Individual bands, which are not all visible on this picture though, were designated ‘a’ to ‘h’. Upon semi-dry electroblotting on a PVDF membrane, each of these bands, except for ‘h’ were shown to share the common N-terminus of chicken POMC.Legend: Lane 1: MM standards (Sigma); lanes 2 – 5: fractions RP-3 to RP-6; lane 6: MM standards.

Fraction

Experimental masses

RP-3 RP-4 RP-5 RP-6 Sequence Theoretical Mass

6950 6950 6950 S1-F63 6954

7007 7008 7008 7008 S1-G64 7011

9535 9596/9758

12 988/13 150

S1-G72 7811

S1-G105 11 178

Ser (2.0) Ser (2.4) Ser (1.5) Ser (9.0) Ser (4.0) Ser (5.0) Ser (1.0) no seq

a b c d e f g h

Gly Gly Gly Gly Gly Gly Gly

Gly 2 (1.8) (2.2) (1.3) (8.9) (4.2) (4.8) (0.9)

Pro Pro Pro Pro Pro Pro Pro

Pro 3 (0.4) (2.7) (1.2) (6.8) (1.4) (3.8) (0.5)

Blnk Blnk Blnk Blnk Blnk Blnk Blnk

Cys 4a Trp Trp Trp Trp Trp Trp Trp

Trp 5 (1.4) (1.6) (0.8) (2.6) (0.6) (1.8) (1.0)

Glu Glu Glu Glu Glu Glu Glu

Glu 6 (0.3) (0.8) (0.9) (3.2) (1.1) (2.0) (0.2) Asn Asn Asn Asn Asn Asn

Asn 7

(0.6) (0.3) (3.7) (0.5) (1.9) (0.3)

Lys (0.2) Lys (1.8) Lys (0.4)

Ser (1.2) Ser (0.4)

Lys 9

Ser (0.1)

Ser 8

(Blnk)

(Blnk)

(Blnk)

Cys 10a

Gln (3.9)

Gln 11

Asp (1.9)

Asp 12

Leu (3.2)

Leu 13

Ala (2.0)

Ala 14

Thr(1.9)

Thr 15

a

Bands are numbered from a to h according to their electrophoretic mobility as shown in Fig. 3, with sample a being the one which migrated the farthest in the gel. Blank cycles such as 4 and 10 are consistent with the presence of a Cys-residue: unmodified cysteines are broken down during the Edman cycle, and do not show up in the HPLC chromatogram. Depending on the amount of protein present on the blot a varying number of residues can be assigned.

Ser 1

POMC Band c /cycle

Table 2 N-terminal sequences of the respective isoforms

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Fig. 4. HP gel permeation chromatogram of fraction RP-4 (lane 3 on Fig. 3) showing the purification of two out of the three major species of the mixture. The third species (7K) was present predominantly in fraction RP-5 (lane 4 in Fig. 3).

human, 59% with bovine, 49% and 38% with the fish sequences. Highest similarities are encountered between the MSH-encoding sequences. Note that the displayed fish POMCs do not contain a g-MSH-like peptide. Interestingly, all tetrapod sequences seem to share a putative N-glycosylation site corresponding to Asn-67 (N). Chicken ACTH shows more resemblance to its homologue in Xenopus (92%) than to ACTH of ostrich (82%).

Fig. 5. Electrophoretic analysis of the HP gel permeation chromatogram. In spite of a 25% difference in apparent molecular weight, these molecules initially co-eluted in RP-HPLC. Lane 1–5: GPC fractions 1 – 5; lane 6: MM standards

4. Discussion Immunization of mice with a chicken pituitary glycoprotein mixture in combination with an immunocytochemical screening protocol, has yielded MAbs with different specificities, including antibodies recognizing the somatotropes (Berghman et al., 1988), the gonadotropes (Berghman et al., 1993) and as described in the present study, the corticotropes. Staining of the chicken corticotropes suggested that the MAb was directed towards a POMC derivative. However, staining in the rat pars intermedia and absence of an immunocytochemical signal in the pars distalis suggested that the antigen recognized was not ACTH, but rather a peptide processed exclusively in the pars intermedia and not in the pars distalis. Since prohormone convertase (PC) 1 but not PC2 is present in the corticotropes and since both PC1 and PC2 are present in the melanotropes, proteolysis is more extensive in the pars intermedia, resulting in a series of smaller peptides including the g-MSHs, CLIP, a-MSH, b-MSH and b-END (Bertagna, 1994) but possibly also in several N-terminal peptides (Van Strien et al., 1995) A straightforward answer to this question was provided by MAb-based immunoaffinity chromatography, by which, in a single step protocol, a preparation suitable for N-terminal sequence determination was produced. Although this preparation was chemically highly heterogeneous as shown by RP-HPLC and SDSPAGE, its N-terminus was homogeneous and suggested that the antigen consisted of a series of N-terminal POMC-derived peptides. This hypothesis was proven

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Table 3 Amino acid and nucleotide sequence of chicken NT-POMC

by showing the presence of at least five identical N-terminal residues in each of the seven bands resolved by SDS-PAGE and individually excised from the PVDF blot. As shown before, the criterium used by monoclonal immunoaffinity, i.e. the occurrence of a highly specific epitope, is able to select for biologically significant mixtures of highly related molecules, which is in contrast to physicochemical purification methods using MM or isoelectric point as the separation criterion, the

latter being of little biological significance for the nature of the retained molecules. Upon specific proteolytic digestions, internal sequence information was obtained which allowed us to reconstruct the protein sequence of the complete N-terminal POMC sequence up to the Glu residue at position 95, leaving a gap of 12 unknown residues before the N-terminus of ACTH. This gap was eventually filled by the nucleotide sequence analyses. Dibasic pairs

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Table 4 Overview of the peptide fragments used for reconstruction of the protein sequence of Table 3 Peptide

Sequence

Mass (theoretical)

Mass (experimental)

Peptide

Sequence

Mass (Theor)

Mass (Exper.)

Kc10 Kc12a Kc12b Kc12c Kc12d Kc13a Kc13b Kc13c Kc13d Kc14a Kc14b Kc14c Kc14d

Ser1 – Lys9 Cys10 – Lys24 Arg75 – Glu95 Tyr53 – Lys62 Ser1 – Lys9 Ala25 – Lys52 Cys10 – Lys24 Tyr53 – Lys62 Ser1 – Lys9 Tyr53 – Lys62 Cys10 – Lys24 Ser1 – Lys9 Ala25 – Lys52

1007.09 1492.73 2116.27 1367.59 1007.09 2994 1492.73 1367.59 1007.09 1367.59 1492.73 1007.09 3711.98

1009.6 1490.67 n/d n/d n/d 4000.01a n/d n/d 4000.01a 1368.6 n/d n/d n/d

Sa6a Sa6b Sa8a Sa8b Sa9a Sa9b Sa9c Sa9d Sa11

Ser49 – Ser56 His57 – Gly72 Ser1 – Glu6 Asn7 – Glu16 Ala17 – Glu29 Ser1 – Glu16 Asn7 – Glu16 Ser49 – Ser56 Val78 – Pro90

983.20 1893.05 677.73 1108.19 1262.52 1767.91 1108.19 983.20 1200.40

982.4 n/d 1782.75a 1782.75a n/d 1766.9 n/d n/d 2866.1

Sa7

His57 – Glu76

2462.7

3676.9 4220.1 4382.0 1979

Leu30 – Glu48 a

The peptides K13a – K13d and Sa8a–Sa8b are linked via disulfide bridges. n/d, not detected.

occur at positions 51 – 52, 65 – 66, 74 – 75 and finally 106 – 107, suggesting the existence of the g-MSHs in chicken (with g3-MSH corresponding to Tyr53 – Arg73 and g-MSH equal to Tyr53-Gly64). The functionally important heptapeptide sequence MSHFRWN is found at positions 55–61 and shows considerable conservation when compared to other POMC homologues. Another aspect of sequence similarity is represented by the conservation of the disulfide bridges. Evidence for disulfide bridge formation between Ala25 – Lys52 and Ser1 –Lys9 is suggested by our finding of a peptide with an experimental MM of 4000 Da, the computed MM of the disulfide-linked peptides being 3998 Da. As reported earlier for ostrich N-terminal POMC (Naude´ et al., 1993), chicken POMC is different from all other isolated POMC N-terminal fragments in that

Fig. 6. Electrophoretic analysis of the apparent MM shift caused by enzymatic digestion with PNGase F. Both major bands undergo a similar shift in the order of 2.5 kDa upon removal of their N-linked carbohydrates. Legend: Lane 1: MM standards (Sigma); lane 2: RP-4 prior to enzymatic digestion; lane 3: the same preparation upon a 24 h digestion with PNGase F.

it is two amino acids longer because of the occurrence of a Ser–Gly sequence at its amino terminus. As in ostrich, the position of the normally occurring Gln or Trp residues is occupied by a Pro residue, forcing the signal peptidase responsible for the removal of the signal peptide to choose an alternative processing site to generate a functional POMC precursor. Further comparison with ostrich POMC revealed the occurrence of the carboxy-terminal Phe–Gly residues that are present within g-MSH in all other tetrapod POMCs, but of which, however, the Gly was missing in ostrich. Naude´ et al. (1993) hypothesized that the pair of dibasic amino acids (Arg65–Arg66) was also present in the ostrich and served as a processing site recognized by prohormone convertases. Our data for chicken clearly corroborate this assumption although we have not found a peptide with a carboxy terminal Phe residue in the a-amidated form, postulated by the above authors. As in all tetrapod POMCs described to date, an N-glycosylation consensus sequence is also found in the chicken at positions 67–69 (Asn67–Ser68–Ser69). Direct evidence for N-glycosylation is provided in the present study by the PNGase F deglycosylation study, showing that two of the three major molecular forms of NT-POMC are indeed glycosylated, since their apparent MM shifts with 2500 Da upon enzymatic cleavage. Accordingly, among the proteolytic products resulting from the Glu-C digestion, we found the peptide His57– Glu76 to be a microheterogeneous glycopeptide with experimental MM of 3677, 4220 and 4382 Da whereas the theoretical MM accounted for by its mere amino acid sequence is only 2462.7 Da. The value of 3676.9 Da can be explained by postulating an Asn-linked glycosylation consisting of GlcNac-GlcNac-Man branched into two mannose residues, i.e. the common pentasaccharide core Man3GlcNac2 extended with two

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Table 5 Sequence similarity of chicken POMC with other vertebrate POMCs.

Legend: Sequence comparison (PCGENE, CLUSTAL) of different POMCs (N-terminal part): POMC – CHICK: Gallus domesticus (chicken); POMC – OSTRI: Struthio camelus (ostrich); COLI – XENLA: (POMC-1) Xenopus lae6is (African clawed frog); COLJ – XENLA: (POMC-2) Xenopus lea6is (African clawed frog); COLI – RANCA: Rana catesbeiana (bull frog); COLI – RANRI: Rana ridibunda (laughing frog) (marsh frog); COLI – BOVIN: Bos taurus (bovine) COLI – MOUSE: Mus musculus (mouse); COLI – HUMAN: Homo sapiens (human); COLI – ONCMY: (POMC-1) Oncorhynchus mykiss (rainbow trout) (Salmo gairdneri); COLJ – ONCMY: (POMC-2) Oncorhynchus mykiss (rainbow trout) (Salmo gairdneri); COLI – ONCKE: Oncorhynchus keta (chum salmon). Characters marked with ‘‘*’’ indicate perfectly conserved residues; characters marked with ‘‘.’’ indicate well conserved residues. ‘‘ – ’’ indicates a deletion.

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Fig. 7. Diagramatic representation of the N-terminal chicken POMC processing. As yet, no evidence was found for O-glycosylation on Ser47. The Ser1–Ile50 fragment which could theoretically result from cleavage at the Arg51 – Lys52 dibasic pair, was present in minute amounts in the immunoaffinity-purified preparation of N-terminal chicken POMC fragments.

more hexoses. The MM of 4220 and 4382 Da still remain to be explained, but it is striking that they differ by the weight of one hexose residue. As expected, the MM of the carbohydrate moieties present on the 11 and 15 kDa forms, are slightly overestimated by the SDSPAGE analysis upon deglycosylation. Detailed mass spectrometric studies of the N-linked oligosaccharides have been conducted on bovine N-terminal POMC by Siciliano et al. (1993). However, none of the structures described in these studies were able to explain the MM of the carbohydrate structures described in the present study for chicken POMC. The absence of any evidence for O-glycosylation on Ser-46 in chicken is also in contrast with what has been described for bovine N-terminal POMC (Siciliano et al., 1994). This lack of O-glycosylation has also been described in ostrich by Naude´ et al. (1993), suggesting that this might be a typical feature of avian POMCs. An important implication of our finding that the apparent absence of O-glycosylation clearly does not result in extensive processing at the dibasic pair Arg51– Lys52, shown by the presence of 6 unprocessed peptides (Fig. 3), is that the chicken corticotroph must display very low PC2 activity. If, indeed, PC2 had displayed significant enzymatic activity we should have found either significant amounts of the peptide Ser1 – Ile50 (if

the epitope recognized by Mab 16D9 occurs on Ser1– Ile50) or a considerable amount of a peptide with Tyr53 as the N-terminus (if the epitope is located more toward the C-terminal end of POMC). On the other hand, we did identify one peptide (band ‘a’ in Fig. 3) which was still smaller than Ser1–Gly64 and which displayed the N-terminus of POMC as shown by Edman degradation on the excised blotting membrane. Although its apparent MM is in the order of 6 kDa, this might be an overestimation and this peptide, though present in minute quantities, might correspond to Ser1–Ile50. This assumption would also be in line with the reported occurrence of small quantities of a-MSH (Hayashi and Imai, 1991) in the chicken pars distalis, which obviously necessitates, at least, some PC2 activity. So far, the combination of C-terminal truncation at dibasic pairs and glycosylation explains a series of isoforms with molecular masses of (1) 7011 Da, (2) 7811 Da, (3) 9026/9596/9758 Da (9 hexose, the N-glycosylated form of (2)), (4) 11178 Da and (5) 12393/12988/ 13150 Da (9hexose, the N-glycosylated form of (4)). The diagram in Fig. 7 shows a pictural summary of these possibilities. Paradoxically, no obvious biological activity can be indicated for this family of peptides, which not only shows extensive polymorphism but also seems to be present in important amounts in the chicken pituitary. Preliminary experiments have shown that

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chicken N-terminal POMC is devoid of corticosteronereleasing activity, nor have we been able to show any lipolytic activity (results not shown), the latter being in contrast to the weak lipolytic activity described for ostrich NT-POMC 1 – 63 (Naude´ et al., 1993). In the literature, a vast number of other biological activities have been described, including mitogenic activity in the adrenal cortex (Lowry et al., 1983; Estivariz et al., 1988a,b), paracrine trophic activity towards the lactotropes in the pituitary (Tilemans et al., 1994; Van Bael et al., 1996), cardioexcitatory actions (Van Bergen et al., 1995) and prolactin-release inhibiting effects in the hypothalamus (Schally et al., 1991). In chicken, however, further research is needed to elucidate the functional role of this complicated peptide family.

Acknowledgements L.R.B., P.V. and J.Vd.B. are senior research associates of the Flemish Fund for Scientific Research (FWOVlaanderen). This work was also supported by a grant from the Flemish Insitute for the Advancement of Scientific and Technological Research in the Industry to H.G. The present study was financially supported by grant G.0259.96 of the Flemish Fund for Scientific Research (FWO-Vlaanderen) to L.R.B. and P.V. and by grant 120.522.93 of a Concerted Research Action of the Flemisch Government. The interaction between F.V. and H.V. was financially supported by grant No. I 98.003 of the Scientific Exchange Program between the Flemish Community and France. We are indebted to Luc Grauwels for his skilful technical assistance and to Julie Puttemans and Marijke Adriaens for photographic assistance.

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