Molecular characterization of two human autoantigens: unique cDNAs encoding 95- and 160-kD proteins of a putative family in the Golgi complex.

Abstract

Sllmmal~Serum autoantibodies from a patient with autoantibodies directed against the Golgi complex were used to screen clones from a HepG2 XZ_ap cDNA library. Three related clones, designated SY2, SY10, and SYll, encoding two distinct polypeptides were purified for further analysis. Antibodies affinity purified by adsorption to the XZ_ap-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 SYll 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 SYll represented 58.7-and 70-kD proteins of 568 and 620 amino acids. The in vitro translation products of SY2 and SYll 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 SYll 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.

A Phosphorylated Cytoplasmic Autoantigen, GW182, Associates with a Unique Population of Human mRNAs within Novel Cytoplasmic Speckles

“…Indirect Immunofluorescence (IIF) analyses used commercially prepared HEp-2 cells (ImmunoConcepts, Sacramento, CA) with fluorescein-conjugated goat anti-human antibodies as previously described (Fritzler et al, 1993). For colocalization studies primary antibodies to the following proteins were used: golgin-97 (murine monoclonal cdf4; Griffith et al, 1997), p58 (murine monoclonal, a gift from Dr. Tom Hobman, University of Alberta), TGN38 (murine monoclonal; Affinity Bioreagents, Golden, CO), clathrin heavy chain (murine monoclonal; Transduction Laboratories, Lexington, KY), rab9 (murine monoclonal, Affinity Bioreagents, Golden, CO), PMP70 (murine monoclonal; Zymed, San Francisco, CA), LAMP1 (murine monoclonal; Drs.…”

Nuclear Aggresomes Form by Fusion of PML-associated Aggregates

“…Aggregates GCP170, also known as golgin-160, was identified as a human auto-antigen in patients with Sjö gren syndrome (Fritzler et al, 1993). Patient sera reacted with an antigen localized in the Golgi, and subsequent studies led to the cloning of GCP170 (Misumi et al, 1997).…”

Autoantigen Golgin-97, an Effector of Arl1 GTPase, Participates in Traffic from the Endosome to theTrans-Golgi Network

“…Most Golgins are autoantigens and were cloned by screening expression libraries with their autoantibodies, such as Golgin-160 (Fritzler et al, 1993), Giantin (Seelig et al, 1994), GMAP-210 (Rios et al, 1994), Golgin-245/p230 (Fritzler et al, 1995), Golgin-97 (Griffith et al, 1997), and Golgin-67 (Eystathioy et al, 2000). Among them, three Golgins, namely, GM130, Giantin, and p115, are the most-well studied.…”