cDNA cloning of a novel human ubiquitin carrier protein. An antigenic domain specifically recognized by endemic pemphigus foliaceus autoantibodies is encoded in a secondary reading frame of this human epidermal transcript.
Autoantibodies from a patient suffering from endemic pemphigus foliaceus (EPF), a blistering skin disease, were used to screen a lambda gt11 human keratinocyte cDNA library. One immunoreactive cDNA clone (lambda EPF5) containing a 900-base pair insert was isolated and subjected to further analysis. Eight of 25 EPF sera were shown to react with the EPF5 fusion protein on immunoblots. The EPF5 cDNA insert hybridized with a 1.2-kilobase epidermal RNA transcript on a Northern blot. Sequence analysis revealed that lambda EPF5 contained the complete coding sequence for a 24-kDa polypeptide exhibiting significant sequence homology with a family of enzymes known as ubiquitin carrier proteins, or E2s, which are an essential component of the ubiquitin-protein conjugation system. The homology was particularly high in the core region containing the active site cysteine. The keratinocyte ubiquitin carrier protein expressed in bacteria, and isolated either intact or as a glutathione S-transferase fusion protein, exhibited the ability to form a thiol ester linkage with ubiquitin in a ubiquitin activating enzyme (E1)-dependent manner, a characteristic property of ubiquitin carrier proteins. The E2 enzyme encoded by clone EPF5 is the first member of this protein family to be cloned from an epidermal source. Interestingly, the EPF autoantibody-reactive epitope and the ubiquitin carrier protein were shown to be encoded in two different translational reading frames. The relevance of the cloned EPF epitope in the pathogenesis of this autoimmune disorder remains to be determined.
Liu
Z
Z
Department of Dermatology, Medical College of Wisconsin, Milwaukee.
Diaz
L A
LA
Haas
A L
AL
Giudice
G J
GJ
D000595:Amino Acid Sequence; D001323:Autoantibodies / Q000378:metabolism*; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification; D015723:Gene Library; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008025:Ligases; D008969:Molecular Sequence Data; D010391:Pemphigoid, Bullous / Q000276:immunology*; D010392:Pemphigus / Q000276:immunology*; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D016364:Reading Frames; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D012016:Reference Values; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic; D044763:Ubiquitin-Conjugating Enzymes; D014452:Ubiquitins / Q000378:metabolism
D001323:Autoantibodies; D002352:Carrier Proteins; D011993:Recombinant Fusion Proteins; D014452:Ubiquitins; C046288:ubiquitin carrier proteins; D012313:RNA; D004247:DNA; D044763:Ubiquitin-Conjugating Enzymes; D005982:Glutathione Transferase; D008025:Ligases