Cloning of candidate autoantigen carboxypeptidase H from a human islet library: sequence identity with human brain CPH.
A number of proteins, many of them enzymes, i.e. glutamic acid decarboxylase (GAD), carboxypeptidase H, 37-40 K tyrosine phosphatase (ICA512, IA2/IA2 beta), have been proposed as islet autoantigens involved in the pathogenesis of IDDM. Until recently, progress in their characterization has been impeded by the inaccessibility of the human pancreas, resulting in many of them being cloned from animal or non-islet sources. Carboxypeptidase H, one of these enzymes, has been cloned and sequenced from human brain and from rat islets but not from human islets. In this study, we describe the production of a human islet cDNA library and the cloning of islet CPH from it. Since CPH clones were also detected in a human thyroid library, we have sequenced CPH from these two endocrine tissue libraries and compared them to the known brain sequence. The sequences from islets and thyroid were identical and differed from brain only in the absence of a second ATG in the predicted 5'non-coding region. Northern blot analysis revealed the presence of an identical 2.5 kb transcript in human islets, thyroid and brain. The confirmation of the existence of a single isoform of CPH expressed in brain and endocrine tissues simplifies future experiments to elucidate the role of CPH as autoantigen.
Alcalde
L
L
IRIBHN, Université Libre de Bruxelles, Hôspital Erasme, Belgium.
Tonacchera
M
M
Costagliola
S
S
Jaraquemada
D
D
Pujol-Borrell
R
R
Ludgate
M
M
D000818:Animals; D000911:Antibodies, Monoclonal; D001324:Autoantigens / Q000235:genetics*; D015152:Blotting, Northern; D043423:Carboxypeptidase H; D002268:Carboxypeptidases / Q000235:genetics / Q000276:immunology*; D002999:Clone Cells; D003001:Cloning, Molecular; D015342:DNA Probes / Q000032:analysis; D003922:Diabetes Mellitus, Type 1 / Q000235:genetics; D015723:Gene Library; D005968:Glutamate Decarboxylase / Q000235:genetics; D006801:Humans; D007328:Insulin / Q000235:genetics; D051381:Rats; D015183:Restriction Mapping; D017421:Sequence Analysis; D013961:Thyroid Gland / Q000378:metabolism
D000911:Antibodies, Monoclonal; D001324:Autoantigens; D015342:DNA Probes; D007328:Insulin; D002268:Carboxypeptidases; D043423:Carboxypeptidase H; D005968:Glutamate Decarboxylase