Molecular characterization of two human autoantigens: unique cDNAs encoding 95- and 160-kD proteins of a putative family in the Golgi complex.
Serum autoantibodies from a patient with autoantibodies directed against the Golgi complex were used to screen clones from a HepG2 lambda Zap cDNA library. Three related clones, designated SY2, SY10, and SY11, encoding two distinct polypeptides were purified for further analysis. Antibodies affinity purified by adsorption to the lambda Zap-cloned recombinant proteins and antibodies from NZW rabbits immunized with purified recombinant proteins reproduced Golgi staining and bound two different proteins, 95 and 160 kD, from whole cell extracts. The SY11 protein was provisionally named golgin-95 and the SY2/SY10 protein was named golgin-160. The deduced amino acid sequence of the cDNA clone of SY2 and SY11 represented 58.7- and 70-kD proteins of 568 and 620 amino acids. The in vitro translation products of SY2 and SY11 cDNAs migrated in SDS-PAGE at 65 and 95 kD, respectively. The in vitro translated proteins were immunoprecipitated by human anti-Golgi serum or immune rabbit serum, but not by normal human serum or preimmune rabbit serum. Features of the cDNA suggested that SY11 was a full-length clone encoding golgin-95 but SY2 and SY10 together encoded a partial sequence of golgin-160. Analysis of the SY11 recombinant protein identified a leucine zipper spanning positions 419-455, a glutamic acid-rich tract spanning positions 322-333, and a proline-rich tract spanning positions 67-73. A search of the SwissProt data bank indicated sequence similarity of SY11 to human restin, the heavy chain of kinesin, and the heavy chain of myosin. SY2 shared sequence similarity with the heavy chain of myosin, the USO1 transport protein from yeast, and the 150-kD cytoplasmic dynein-associated polypeptide. Sequence analysis demonstrated that golgin-95 and golgin-160 share 43% sequence similarity and, therefore, may be functionally related proteins.
Fritzler
M J
MJ
W. M. Keck Autoimmune Disease Center, Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, California 92037.
Hamel
J C
JC
Ochs
R L
RL
Chan
E K
EK
D000595:Amino Acid Sequence; D000818:Animals; D001324:Autoantigens / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D005260:Female; D005455:Fluorescent Antibody Technique; D006056:Golgi Apparatus / Q000276:immunology*; D000077614:Golgi Matrix Proteins; D006801:Humans; D015151:Immunoblotting; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D014176:Protein Biosynthesis; D011506:Proteins; D011817:Rabbits; D011994:Recombinant Proteins / Q000276:immunology
D001324:Autoantigens; C108388:GOLGA3 protein, human; D000077614:Golgi Matrix Proteins; C097741:Golgin subfamily A member 2; D008565:Membrane Proteins; D011506:Proteins; D011994:Recombinant Proteins; D004247:DNA