Molecular cloning and ultrastructural localization of the core protein of an eggshell matrix proteoglycan, ovocleidin-116.
The role of avian eggshell matrix proteins in shell formation is poorly understood. This calcitic biomaterial forms in a uterine fluid where the protein composition varies during the initial, calcification, and terminal phases of eggshell deposition. A specific antibody was raised to a 116-kDa protein, which is most abundant in uterine fluid during active eggshell calcification. This antiserum was used to expression screen a bacteriophage cDNA library prepared using mRNA extracted from pooled uterine tissue harvested at the midpoint of eggshell calcification. Plasmids containing inserts of differing 5'-lengths were isolated with a maximum cDNA sequence of 2.4 kilobases. Northern blotting and reverse transcriptase-polymerase chain reaction demonstrated that the 2. 35-kilobase message was expressed in a uterine-specific manner. The hypothetical translational product from the open reading frame corresponded to a novel 80-kDa protein, which we have named ovocleidin-116. After removal of the predicted signal peptide, its N-terminal sequence corresponded almost exactly with that determined from direct microsequencing of the 116-kDa uterine protein (this work) and with that previously determined for the core protein of a 120-kDa eggshell dermatan sulfate proteoglycan (Corrino, D. A., Rodriguez, J. P., and Caplan, A. I. (1997) Connect. Tissue Res. 36, 175-193). Ultrastructural colloidal gold immunocytochemistry of ovocleidin-116 demonstrated its presence in the organic matrix, in small vesicles found throughout the mineralized palisade layer, and the calcium reserve assembly of the mammillary layer. Ovocleidin-116 thus is a candidate molecule for the regulation of calcite growth during eggshell calcification.
Hincke
M T
MT
Department of Cellular Medicine, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada. mhincke@uottawa.ca
Gautron
J
J
Tsang
C P
CP
McKee
M D
MD
Nys
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D015153:Blotting, Western; D002113:Calcification, Physiologic; D002119:Calcium Carbonate / Q000737:chemistry; D002645:Chickens; D003001:Cloning, Molecular; D003871:Dermatan Sulfate / Q000737:chemistry; D004527:Egg Proteins / Q000737:chemistry / Q000235:genetics*; D016326:Extracellular Matrix Proteins / Q000737:chemistry; D005260:Female; D007150:Immunohistochemistry; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D011509:Proteoglycans / Q000737:chemistry*; D012333:RNA, Messenger / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D014599:Uterus / Q000737:chemistry
D004527:Egg Proteins; D016326:Extracellular Matrix Proteins; D011509:Proteoglycans; D012333:RNA, Messenger; C402224:ovocleidin-116; D003871:Dermatan Sulfate; D002119:Calcium Carbonate