Wmuws, D0CT0R 0F PHIL0S0PHY

PRECURSOR AND GENE STRUCTURE OP A GROWTH HORMONE-RELEASING HORMONE-LIKE MOLECULE AND PITUITARY ADENYLATE CYCLASE ACTIVATING POLYPEPTIDE FROM SOCKEYE SALMON BRAIN David B . Parker B.Sc., Simon Fraser University, 1978 M.Sc., Simon Fraser University, 1985 h Dissertation submitted in Partial Fulfillment of the Requirements for the Degree of ACCRI'TEO W i i l t y wmuws, D0CT0R 0F PHIL0S0PHY at*.- We accept this thesis as conforming . y AN to the required standard J A** A m f \ * ' ” *" ite 'fe stLAp JC& l Dr, N.M. Sherwood/ Supervisor (Dept, of Biology) Dr, G .0. Macjcie, Departmj^fTtal Member (Dept, of Biology) — — ----- v — — -- Dr. R.D. Burkji,.departmental Member (Dept, of Biology) D r , T.W.^ Peairson, Outside Member (Dept, of Biochemistry) ~ r - t - ----------------- -— 1— ----------- ------------- --------------- ---------------- - ———-------- - Dr. C.Jj. Hew, CExternal Examiner) ©DAVID B. PARKER, 1992 University of Victoria All rights reserved. This dissertation may not be reproduced in whole or part, by photocopying or other means, without permission of the author II Supervisor? Dr. Nancy M. Sherwood ABSTRACT Growth Hormone-releasing hormone (GHRH) is a neuropeptide which stimulates the synthesis and release of growth hormone (GH) from the pituitary gland. The primary structure of this peptide has been identified in 7 mammalian species while the gene has been isolated from only rat and human. GHRH is a member of the glucagon superfamily which includes vasoactive intestinal peptide (VXP), glucagon, secretin, peptide histidine methionine (PHM), gastric inhibitory peptide (GXP) and a recently identified peptide, pituitary adenylate cyclase activating polypeptide (PACAP). The evolutionary relationships of this superfamily are not well understood because the gene structure of these molecules has only been identified in mammals. This thesis presents immunological evidence of a GHRH-like molecule, and identifies a GHRH/PACAP precursor and gene that encode two peptides, a GHRH-like molecule structurally related to PACAP-related peptide (PRP) and PACAP, from sockeye salmon brain. An antiserum directed against a topologically assembled epitope Of human GHRH 1-44 (NH2) was produced and used to develop a radioimmunoassay for detection of immunoreactive GHRH in brain extracts of salmon, guinea pig, mouse and alligator. An immunoreactive GHRH from salmon brain extracts with a retention time on reverse phase high*' performance liquid chromatography (HPLC) distinct from human GHRH was present. In alligator, the same antiserum also detected a GHRH-like molecule. During attempts to purify alligator GHRH, alligator brain neuropeptide Y (NPY) was identified. Alligator NPY is the first non-mammalian vertebrate to have 100% sequence identity to human NPY. The sequence identity between alligator and human NPY suggests that this sequence is the same as the ancestral amniote NPY. Molecular biological techniques were used for the structural identification of the salmon GHRH-like molecule and another peptide. The salmon GHRH/PACAP precursor contains 173 amino acids and has dibasic and monobasic processing sites for cleavage of a 45 amino acid GHRH-like peptide with a free acid C-terminus and a 38 amino acid PACAP with an amidated C-terminus. The salmon GHRH-like peptide has 40% amino acid sequence identity with the human GHRH and 56% identity with human PACAP-related peptide (PRP). salmon PACAF-38 is highly conserved (89%) with only 4 amino acid substitutions compared with the human, ovine ana rat PACAP-38 peptides. Nucleotide sequenc.ing and use of the polymerase chain reaction show the exon/intron organization of the salmon GHRH/PACAP gene to be similar to the human PACAP gene. Unlike the mammalian PACAP genes, the salmon gene produces two precursor forms by post-transcriptional processing. One IV form is similar to the mammalian PACAP precursors, while the second form is shorter due to the excision of exon 4. This deletion results in the loss of the first 32 amino acids of the GHRH-like peptide from the precursor. The high sequence identity and structural organization between the GHRH(PRP)/PACAP and PHM(PHI)/VIP genes suggest a duplication event occurred in an ancestral gene after the divergence from the other glucagon superfamii.y members. GHRH in mammals may have arisen by gene duplication after the divergence of the tetrapods from the other vertebrate lines. Thus, GH in fish may be controlled by the two molecules, GHRH-like peptide and PACAP, located on a single GHRH/PACAP gene. Examiners: <» ---- — ---- y. ............... — --- '----- ----- ----- Dr. N.M. Sherwood, Supervisor (Dept, of Biology) Dr. G .0./ Mackie, Departmental Member (Dept, of Biology) ---- f— \y— -----p?------- -— ——— Dr. R.D. BurKe,«<D£ipartmental Member (Dept, of Biology) Dr. T . W . P e a r L o n , O u t s i d e Member (Dept, of Biochemistry) ■ ■ Dr. C.L. Hew, External Examiner, (Depts. of Clinical Biochemistry and Biochemistry, University of Toronto) V TABLE OF CONTENTS ABSTRACT. .............................. .i.i TABLE OF CONTENTS . .............................. v LIST OF TABLES........... vii LIST OF FIGURES. .............................. .viii LIST OF ABBREVIATIONS. ....... xi ACKNOWLEDGEMENTS. ...... .xii CHAPTER u General Introduction. ..... 1 CHAPTER 2: Evidence for a growth hormone-releasing hormone like molecule in salmon brain, Onaorhynchus kata and 0 . kisutch ..... ...... , . 35 Introduction 3 6 Methods and Materials .......... 38 Results,...... 45 Discussion.................... 66 CHAPTER 3: Primary structure of neuropeptide Y from brains of the American alligator (Alligator mississipiensis) ..... 69 Introduction ..*70 Methods and Materials. ...... 71 Results.. c ...«/6 Discussion............. ............... .......89 CHAPTER 4: Two Salmon neuropeptides coded in one brain oDNA are structurally related to growth hormone- releasing hormone (GHRH) and pituitary adenylate cyclase activating polypeptide (PACAP) . ..... 93 Introduction ........... 94 Methods and Materials ...... 97 Results ..... 103 Discussion.. .......... 135 CHAPTER 5: Gene sequence of sockeye salmon GHRH-like and PACAP molecules; Evolutionary relationship to the glucagon superfamily. ........ 151 Introduction .; i............. 152 Methods and Materials ..... 153 Results .... ........ ................157 Discussion.............a............................185 CHAPTER 6: General Discussion....... .192 Evolution of the glucagon superfamily. ...... 193 Evolution of the PACAP ainu VIP genes 197 VI The possibility of another GHRH gene without PACAP in salmon...................... .205 Structure/function relationships in the evolution of the PACAP and VIP genes............................... .206 The function of the salmon GHRH/PACAP gene.......... 209 Future Directions......................... 211 literature cited ........................ 213 LIST OF TABLES Table l.l Growth hormone-releasing hormones.........,.....8 Table 2.1 Cross-Reactivity of Ovine and Rat GHRH with hGBRH 1-44 using Antiserum 8119 (1:45,000)........... 51 Table 3.1 Yield of phenylthiohydantoin (PTH)-derivatized amino acids ....... 78 Table 6.1 A comparison of the exon encoding the salmon GHRH-like peptide or human GHRH domains with the respective exons of the human PHM and PRP genes....... 208 VIII LIST OF FIGURES Chapter % Figure 1.1 A schematic diagram showing the interaction of GHRH, SS, and IGF-I on the control of growth hormone release in mammals ..,12 Figure 1,2 A schematic diagram showing the exon/intron organisation of the human growth hormone- releasing hormone gene. (GHRH) and its m R N A . .27 Chapter 2 Figure 2.1 Standard curve of hGHRH 1-44 NH2 using antiserum 8119 at a final dilution of 1:45,000..........52 Figure 2.2 Binding of hGHRH fragments with three antisera developed against hGHRH 1-44 NH2 ......... .54 Figure 2.3 Immunoreactive fractions of coho and chum salmon brain crude extracts after reverse- phase HPLC ..................56 Figure 2.4 Reverse-phase HPLC of pooled fractions of coho or churn irGHRH. ........ 58 Figure 2.5 Reverse-phase HPLC of immunoaffinity purified material from chum salmon brains... ...... 60 Figure 2.6 Reverse-phase HPLC of immunoaffinity purified material from chum salmon brains..............62 Figure 2.7 Reverse-phase HPLC of pooled fractions of chum salmon irGHRH ............ 64 Chapter 3 Figure 3.1 Binding of VIP (human, porcine and rat), PHI (porcine), secretin (chicken) and NPY (human) compared to the hGHRH(1-44 NH2 ) standard curve ............. .................. 79 Figure 3.2 Initial purification strategy of alligator brain immunoreactive GHRH. ..... 81 Figure 3.3 An analytical rpHPLC run of immunoreactive fractions ..... S3 Figure 3.4 Final purification of immunoreactive material on HPLC.._____ ____________________..._______ 85 Figure 3.5 Coelution of alligator NPY with synthetic human NPY and NPY-OH Using rpHPLC....... ...87 IK Chapter Figure 4 . 1 A schematic diagram showing the position of the primers used in the polymerase chain reaction of sockeye salmon cDNA.Ill Figure 4 .2 A schematic diagram of the strategy used in the polymerase chain reaction (PCR) 113 Figure 4 .3 Photograph of sockeye salmon and catfish pcp. amplification products run on a 2% agarose gel.............. 115 Figure 4 .4 Photograph showing a Pvu II digest of miniprep plasmid DNA obtained from cloned PCR products........... *.................... 117 Figure 4 .5 Nucleotide sequence and the corresponding amino acid sequence of the sockeye salmon GHRH/PACAP cDNA............ .119 Figure 4 .6 Nucleotide

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