Expression and localization of leucine-rich B7 protein in human ocular tissues.
OBJECTIVE
Using a web-based tool to screen the human proteome for potential mimecan/osteoglycin interacting proteins, we found that the leucine-rich B7 protein may have functional associations with several small leucine-rich proteoglycans (SLRP), including mimecan. The purpose of this study was to determine the expression of leucine-rich B7 protein in human eye tissues and its subcellular localization in MG-63 cells.


METHODS
Primers were synthesized to amplify the two known differentially spliced B7 mRNA transcripts. Reverse transcription-polymerase chain reaction (RT-PCR) amplification was used to determine the expression of B7 mRNAs in human ocular and nonocular tissues. A rabbit anti-human B7 antibody was generated that specifically immunostained B7 proteins. The expression of B7 proteins in the human eye was determined by immunohistochemistry (IHC). Intracellular localization of leucine-rich B7 and mimecan proteins were determined using transient co-transfections, cell immunostaining, and laser scanning confocal microscopy.


RESULTS
RT-PCR analysis showed moderate expression of B7, transcript variant 2, in human cornea, iris, sclera, and retina. In contrast, B7, transcript variant 1, was strongly expressed only in the cornea. The two B7 mRNAs were highly expressed in human brain, blood, peripheral mononuclear cells, and other human tissues. By IHC, immunostaining for leucine-rich B7 protein was found in epithelial and endothelial layers of the cornea, epithelial and fiber cells of the lens, in sclera, and in the rod and cone layer of the retina of adult human eye. Leucine-rich B7 protein was found to localize to both nucleus and cytoplasm of MG-63 cells, whereas mimecan was found only in the cytoplasm of these cells. Merged images obtained by confocal microscopy revealed certain cytoplasmic regions in MG-63 cells where B7 and mimecan proteins appeared to co-localize.


CONCLUSIONS
The present work is the first to demonstrate the expression and localization of leucine-rich B7 protein in human eye and other human tissues. The results reported here are an essential prerequisite for future studies aimed at understanding the biological roles of leucine-rich B7 proteins in health and disease.
Tasheva
Elena S
ES
Division of Biology, Kansas State University, Manhattan, KS 66506-4901, USA. est@ksu.edu
An
Ke
K
Boyle
Daniel L
DL
Conrad
Gary W
GW
D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D005123:Eye / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression; D006023:Glycoproteins / Q000235:genetics; D006801:Humans; D015151:Immunoblotting; D007124:Immunoenzyme Techniques; D036341:Intercellular Signaling Peptides and Proteins; D018613:Microscopy, Confocal; D020543:Proteome; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012795:Sialoglycoproteins / Q000235:genetics*; D014162:Transfection
D006023:Glycoproteins; D036341:Intercellular Signaling Peptides and Proteins; C504461:LRRC7 protein, human; C063488:OGN protein, human; D020543:Proteome; D012333:RNA, Messenger; D012795:Sialoglycoproteins