Ovocalyxin-32, a novel chicken eggshell matrix protein. isolation, amino acid sequencing, cloning, and immunocytochemical localization.
The eggshell is a highly ordered structure resulting from the deposition of calcium carbonate concomitantly with an organic matrix upon the eggshell membranes. Mineralization takes place in an acellular uterine fluid, which contains the ionic and matrix precursors of the eggshell. We have identified a novel 32-kDa protein, ovocalyxin-32, which is expressed at high levels in the uterine and isthmus regions of the oviduct, and concentrated in the eggshell. Sequencing of peptides derived from the purified protein allowed expressed sequence tag sequences to be identified that were assembled to yield a full-length composite sequence whose conceptual translation product contained the complete amino acid sequence of ovocalyxin-32. Data base searches revealed that ovocalyxin-32 has limited identity (32%) to two unrelated proteins: latexin, a carboxypeptidase inhibitor expressed in the rat cerebral cortex and mast cells, and a skin protein, which is encoded by a retinoic acid receptor-responsive gene, TIG1. High level expression of ovocalyxin-32 was limited to the isthmus and uterus tissue, where immunocytochemistry at the light and electron microscope levels demonstrated that ovocalyxin-32 is secreted by surface epithelial cells. In the eggshell, ovocalyxin-32 localizes to the outer palisade layer, the vertical crystal layer, and the cuticle of the eggshell, in agreement with its demonstration by Western blotting at high levels in the uterine fluid during the termination phase of eggshell formation. Ovocalyxin-32 is therefore identified as a novel protein synthesized in the distal oviduct where hen eggshell formation occurs.
Gautron
J
J
Station de Recherches Avicoles, Institut National de la Recherche Agronomique, Nouzilly, 37380 France.
Hincke
M T
MT
Mann
K
K
Panheleux
M
M
Bain
M
M
McKee
M D
MD
Solomon
S E
SE
Nys
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D000941:Antigens / Q000737:chemistry; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D001842:Bone and Bones / Q000378:metabolism; D002356:Cartilage / Q000378:metabolism; D002642:Chick Embryo; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D019992:Databases as Topic; D004527:Egg Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006046:Gold / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D056892:Mediator Complex; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D010057:Oviducts / Q000648:ultrastructure; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014018:Tissue Distribution; D014157:Transcription Factors / Q000378:metabolism; D014599:Uterus / Q000378:metabolism / Q000648:ultrastructure
D000941:Antigens; D018076:DNA, Complementary; D004527:Egg Proteins; C086812:Lxn protein, rat; C416944:MED15 protein, human; D056892:Mediator Complex; D010455:Peptides; D012333:RNA, Messenger; D014157:Transcription Factors; C439069:ovocalyxin 32; D006046:Gold