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Isolation and Characterization of a C4-Like Complement Protein from Chicken Plasma Woan-Hwa Renee Lee Iowa State University

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Isolation and Characterization of a C4-Like Complement Protein from Chicken Plasma Woan-Hwa Renee Lee Iowa State University Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 1984 Isolation and characterization of a C4-like complement protein from chicken plasma Woan-Hwa Renee Lee Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Agriculture Commons, Poultry or Avian Science Commons, Veterinary Microbiology and Immunobiology Commons, and the Veterinary Physiology Commons Recommended Citation Lee, Woan-Hwa Renee, "Isolation and characterization of a C4-like complement protein from chicken plasma " (1984). Retrospective Theses and Dissertations. 9003. https://lib.dr.iastate.edu/rtd/9003 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. INFORMATION TO USERS This reproduction was made from a copy of a document sent to us for microfilming. While the most advanced technology has been used to photograph and reproduce this document, the quality of the reproduction is heavily dependent upon the quality of the material submitted. The following explanation of techniques is provided to help clarify markings or notations which may appear on this reproduction. 1.The sign or "target" for pages apparently lacking from the document photographed is "Missing Page(s)". If it was possible to obtain the missing page(s) or section, they are spliced into the film along with adjacent pages. This may have necessitated cutting through an image and duplicating adjacent pages to assure complete continuity. 2. When an image on the film is obliterated with a round black mark, it is an indication of either blurred copy because of movement during exposure, duplicate copy, or copyrighted materials that should not have been filmed. For blurred pages, a good image of the page can be found in the adjacent frame. If copyrighted materials were deleted, a target note will appear listing the pages in the adjacent frame. 3. When a map, drawing or chart, etc., is part of the material being photographed, a definite method of "sectioning" the material has been followed. It is customary to begin filming at the upper left hand comer of a large sheet and to continue from left to right in equal sections with small overlaps. If necessary, sectioning is continued again—beginning below the first row and continuing on until complete. 4. For illustrations that cannot be satisfactorily reproduced by xerographic means, photographic prints can be purchased at additional cost and inserted into your xerographic copy. These prints are available upon request from the Dissertations Customer Services Department. 5. Some pages in any document may have indistinct print. In all cases the best available copy has been filmed. UniversiV MicKÏilms International 300 N. Zeeb Road Ann Arbor, Ml 48106 8423648 Lee, Woan-Hwa Renee ISOLATION AND CHARACTERIZATION OF A C4-LIKE COMPLEMENT PROTEIN FROM CHICKEN PLASMA Iowa State University PH.D. 1984 University Microfilms Int6rnsti 0nsi 300 N. Zœb Road, Ann Arbor, Ml 48106 PLEASE NOTE: In all cases this material has been filmed in the best possible way from the available copy. Problems encountered vwth this document have been identified here vwth a check mark 1. Glossy photographs or pages. y 2. Colored illustrations, paper or print ^ 3. Photographs with dark background ^ 4. Illustrations are poor copy 5. Pages with black marks, not original copy. 6. Print shows through as there is text on both sides of page. 7. Indistinct, broken or small print on several pages ^ 8. Print exceeds margin requirements 9. Tightly bound copy with print lost in spine 10. Computer printout pages with indistinct print. 11. Page(s) lacking when material received, and not available from school or author. 12. Page(s) seem to be missing in numbering only as text follows. 13. Two pages numbered . Text follows. 14. Curling and wrinkled pages 15. Other University Microfilms International Isolation and characterization of a C4-like complement protein from chicken plasma by Woan-Hwa Renee Lee A Dissertation Submitted to the Graduate Faculty in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Department: Animal Science Major: Immunobiology Approved: Signature was redacted for privacy. In Charge of Major Voik Signature was redacted for privacy. For the Major Department Signature was redacted for privacy. duate College Iowa State University Ames, Iowa 1984 ii TABLE OF CONTENTS Page GLOSSARY AND ABBREVIATIONS vii INTRODUCTION 1 LITERATURE REVIEW 5 Structure of the Major Histocompatibility Complex (MHC) 3 The Classical Pathway of the Human Complement Cascade Reaction 11 04 as a Cell Surface Antigen 15 Studies of Mouse Serum Substance Protein (Ss) and Sex-limited Protein (Slp) 17 Studies of Chicken Complement 21 MATERIALS AND METHODS 25 Chicken Stocks 25 Complement Activity 28 Silastic Tube Implantation % Affinity Chromatography 38 Rabbit Immunization 39 SDS-Polyacrylamide Gel Electrophoresis (PAGE) 39 Immunofixation 40 Immunoelectrophoresis 41 Radial Immunodiffusion (RID) 41 Immunofluorescent Labeling of Cells 42 Immunoprecipitation 43 EXPERIMENTAL DESIGN AND RESULTS 46 Complement Activity in the Leghorn 81 Population 46 Isolation and Purification of Chicken C4 Complement Component 51 Characterization of Rabbit Anti-chicken C4 53 SDS-polyacrylamide Gel Electrophoresis 58 Kinetic Study of Chicken C4 Protein Levels in the SI Population 62 The C4 Protein Level in Chicken Plasma 62 C4 Protein Levels in Chickens Challenged with Rous Sarcoma Tumor Virus (RSV) 66 04 Protein and Chicken Cell Surface Antigens 68 04 Protein Polymorphisms 72 Sex Differences in C4 Protein Levels 75 ill DISCUSSION 78 Blueprint of Research 78 Purification and Isolation of Chicken C4 79 C4 Level in Normal and Tumor-Developing Chicken 85 C4 as a Cell Surface Antigen 84 C4 Polymorphism Revealed by Immunofixation 89 C4 Level and the Sex Differences 89 CONCLUSIONS 91 SUMMARY 95 BIBLIOGRAPHY 94 ACKNOWLEDGEMENTS 105 iv LIST OF TABLES Page Table 1. Total complement and complement components in domestic animals (CH^^) 23 Table 2. Iowa inbred lines used in the study 26 Table 3. Classification of Iowa State University B haplotypes of lines with respect to Illinois Ea-B reference alleles and to an Edmonton panel of monoclonal B-complex antibodies 29 Table 4. The test tube scheme for the determination of complement titer 32 Table 5. Analysis of variance of complement activity 47 Table 6. Mean complement activity (in CH(-Q) at 3 ages of birds in the SI line 48 Table 7» Cross hemolytic reactions between chicken and human C4 57 Table 8. Analysis of variance of 04 protein levels in chicken plasma collected at different ages 65 2 Table 9. C4 protein (d ) in plasma samples from chickens challenged with Rous sarcoma virus (RSV) 69 Table 10. Cell surface proteins immunoprecipitated by rabbit anti-chicken 04 in B complex haplotypes of the S1 line 71 2 Table 11. 04 protein level (d ) in normal and castrated males 75 Table 12, 04 protein level (d^) in normal and ovarectomized females 77 V LIST OF FIGURES Page Figure 1. Genetic map of the H-2 system 4 Figure 2. Genetic map of the HLA system 7 Figure 3« Hypothetical genetic map of the chicken B complex 10 Figure 4-. The classical pathway of the complement cascade reaction 13 Figure 5* Electrophoretic patterns of human 04 protein taken from the literature 18 Figure 6. Proposed polypeptide chain structure of C4 protein 19 Figure ?• Graphic determination of complement titer of Figure 8. Eadial hemolytic rings 35 Figure 9. Overall complement activity of four MHC haplotypes of the 51 Leghorn line 49 Figure 10. Complement activity by sex in the 81 Leghorn line 50 Figure 11. Cross-reactivity between chicken plasma and antiserum against mammalian 04 52 Figure 12. Immunoelectrophoresis analysis of rabbit antisera 54 Figure 13« Results of hemolysis inhibition test 56 Figure 14» Molecular weight determination of 04 protein in % SDS-PAGE 59 Figure 15» Molecular weight determination of nonreduced and reduced 04 protein on 3% SDS-PAGE 60 Figure 16. Molecular weight determination of reduced 04 protein on 1.5% SDS-PAGE 61 vi Figure 17. Levels of 04 protein in chicken plasma by age groups 65 Figure 18. Levels of 04 protein for four B complex haplotypes from the Si Leghorn line 64 Figure 19» Standard curve for determining 04 protein level in chicken plasma 67 Figure 20. Electrophoretic pattern of 04 protein in barbitol buffer 75 Figure 21. Electrophoretic patterns of 04 protein in tris-glycine-barbitol buffer 74 vii GLOSSARY AND ABBREVIATIONS BT: Blood type; Ea-B is the primary marker and the superscript 1 2 designates the allele, i.e., B , B , etc. 04 protein level: 04 protein concentration, as determined on RID. Two 2 types of units have been used; d and mg/ml. Crude data taken 2 directly from measuring the areas of precipitation rings are in d . Otherwise, data corrected with a 04 protein concentration standard curve are in mg/ml. OH^Q; The amount of complement that can lyse one half of a standard cell number under standard conditions, i.e., the amount of complement which lyses 1.25x10^ sensitized sheep erythrocytes in a total reaction volume of 1.2 ml. d 2 ; square of the diameter of the RID ring; directly proportional to the area. GAT: a synthetic peptide from glutamin^^-alanine^^-tyrosine^^. Ir-GAT: Immune response to GAT. MHO: Major histocompatibility complex; a cluster of genes coding for cell surface antigens which are important in regulating both cellular and humoral immune responses. OCA: Overall complement activity determined as the degree to which sensitized sheep red blood cells are lysed. PBS: Phosphate buffered solution; 55 mM NaHgPO^ HgO/GY mM NagHPO^ 7 HgO/lSS mM NaCl; pH 7.0.
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