Goodpasture Antigen-binding Protein Is a Soluble Exportable Protein That Interacts with Type IV Collagen

Abstract

Goodpasture-antigen binding protein (GPBP) is a nonconventional Ser/Thr kinase for basement membrane type IV collagen.Various studies have questioned these findings and proposed that GPBP serves as transporter of ceramide between the endoplasmic reticulum and the Golgi apparatus. Here we show that cells expressed at least two GPBP isoforms resulting from canonical (77-kDa) and noncanonical (91-kDa) mRNA translation initiation. The 77-kDa polypeptide interacted with type IV collagen and localized as a soluble form in the extracellular compartment. The 91-kDa polypeptide and its derived 120-kDa polypeptide associated with cellular membranes and regulated the extracellular levels of the 77-kDa polypeptide. A short motif containing two phenylalanines in an acidic tract and the 26-residue Ser-rich region were required for efficient 77-kDa polypeptide secretion. Removal of the 26-residue Ser-rich region by alternative exon splicing rendered the protein cytosolic and sensitive to the reduction of sphingomyelin cellular levels. These and previous data implicate GPBPs in a multicompartmental program for protein secretion (i.e. type IV collagen) that includes: 1) phosphorylation and regulation of protein molecular/supramolecular organization and 2) interorganelle ceramide trafficking and regulation of protein cargo transport to the plasma membrane. Goodpasture antigen-binding protein (GPBP)2 phosphorylates the noncollagenous-1 (NC1) domain of the ␣3 chain of type IV collagen (␣3(IV)NC1) (1). This domain is a pivotal structure in the molecular and supramolecular organization of the glomerular basement membrane collagen and also the target of autoantibodies mediating glomerulonephritis in Goodpasture disease (2). Increased GPBP expression has been associated with autoimmune pathogenesis including Goodpasture disease (3) and with the induction of glomerular basement membrane collagen disorganization and deposit of IgA antibodies (4). These observations suggest that GPBP regulates glomerular basement membrane collagen organization and induces type IV collagen-based antibody-mediated glomerulonephritis when its expression is abnormally elevated (3, 4). The GPBP gene (COL4A3BP) also encodes for GPBP⌬26, a more abundant, less active, alternatively spliced GPBP variant lacking a 26-residue Ser-rich region that is apparently not regulated under these pathological conditions (3).GPBP contains multiple structural elements including N-terminal pleckstrin homology domain, Ser-Xaa-Yaa region, bipartite nuclear localization signal, coiled-coil domain, two phenylalanines in an acidic tract (FFAT) motif, and C-terminal steroidogenic acute regulatory related lipid transfer (START) domain. Additional structural features include motifs for selfinteraction and phosphorylation (1, 3, 5, 6). The pleckstrin homology domains comprise a variety of poorly conserved structures present only in eukaryotes that have been proposed to mediate protein targeting to cellular membranes through interaction with phosphoinositides (7). A variety of proteins inc...

Goodpasture Antigen-binding Protein/Ceramide Transporter Binds to Human Serum Amyloid P-Component and Is Present in Brain Amyloid Plaques

“…Antibodies against GPBP epitopes 1-50 ( Fig. 8) and 300 -350 (data not shown) detected a GPBP fragment of ϳ35-37 kDa, which has been described previously (22). This fragment was not present in a pull-down performed with an isotype control antibody when detected with the same GPBPspecific antibodies.…”

“…GPBPs exist in different isoforms that are found in the extracellular compartment, either soluble or associated with the surface of the plasma membrane (21,22), and in blood (23). A shorter splicing isoform, CERT, is located inside the cell and functions as carrier for ceramide from the ER to the Golgi apparatus (24).…”

Human Biliverdin Reductase Suppresses Goodpasture Antigen-binding Protein (GPBP) Kinase Activity

“…Cells express at least two GPBP isoforms of 77 and 91 kDa. The 77-kDa GPBP polypeptide (GPBP in this report) interacts with type IV collagen, whereas the 91-kDa isoform associates with the cellular membrane and regulates extracellular levels of the 77-kDa polypeptide (7). In the present study we concentrated on the interaction of hBVR with the 77-kDa form of GPBP by transfecting cells with an expression plasmid encoding this form and using the cytoplasmic fraction of the cell lysate; hBVR is a soluble protein.…”

Complement Activation by Ceramide Transporter Proteins

“…CERT and CERT L are expressed by a wide range of tissues and cells (20,23), and both extracellular and membrane-bound isoforms of CERT L were described (22). To assess whether endogenous CERT L is involved in the interaction of C1q with apoptotic cells, the binding of purified C1q on etoposide-treated apoptotic Jurkat cells was studied.…”

“…The shorter splicing isoform CERT is ubiquitously present inside the cell and functions as a carrier for ceramide between the endoplasmic reticulum and the Golgi apparatus (19,21). The longer splicing isoform CERT L is a nonconventional serine/threonine kinase that is found in the extracellular compartment either in soluble form or associated with the surface of the plasma membrane (22). Based on the known properties of CERT, we hypothesize that it has a function in the innate immune system.…”