Juan Saus

Characterization of a Novel Type of Serine/Threonine Kinase That Specifically Phosphorylates the Human Goodpasture Antigen

Goodpasture disease is an autoimmune disorder that occurs naturally only in humans. Also exclusive to humans is the phosphorylation process that targets the unique N-terminal region of the Goodpasture antigen. Here we report the molecular cloning of GPBP (Goodpasture antigen-bindingprotein), a previously unknown 624-residue polypeptide. Although the predicted sequence does not meet the conventional structural requirements for a protein kinase, its recombinant counterpart specifically binds to and phosphorylates the exclusive N-terminal region of the human Goodpasture antigen in vitro. This novel kinase is widely expressed in human tissues but shows preferential expression in the histological structures that are targets of common autoimmune responses. The work presented in this report highlights a novel gene to be explored in human autoimmunity.

Cross-talk between different enhancer elements during mitogenic induction of the human stromelysin-1 gene.

Platelet-derived growth factor (PDGF) induces the expression of human stromelysin-1, a matrix metalloproteinase involved in tumor invasion and metastasis. Here it is shown that stromelysin-1 gene induction by PDGF depends on Ras and involves three previously identified promoter elements (the stromelysin-1 PDGF-responsive element (SPRE) site, the two head-to-head polyomavirus enhancer A-binding protein-3 (PEA3) sites, and the activator protein-1 (AP-1) binding site). During mitogenic induction, these responsive elements appear to be organized in two independent transcriptional units, SPRE-AP-1 and PEA3-AP-1, which result from specific element cross-talking. Interestingly, expression of a dominant negative mutant of Raf-1 significantly interfered with the induction through PEA3-AP-1 but not with that operating through SPRE-AP-1. Conversely, only the induction operating through SPRE-AP-1 was affected significantly by the expression of a dominant negative mutant of the atypical λ/ι protein kinase C (λ/ιPKC). These data strongly suggest that the signal triggered by PDGF flows through Ras and bifurcates toward two distinct pathways, one operating through Raf and involving PEA3-AP-1 and the other one Raf-independent, operating through λ/ιPKC and SPRE-AP-1. Furthermore, we present evidence suggesting that the novel SPRE-binding transcription factor SPBP cross-couples with c-Jun to transactivate the SPRE site.

Human Podocytes Adhere to the KRGDS Motif of the α3α4α5 Collagen IV Network

Podocyte adhesion to the glomerular basement membrane is required for proper function of the glomerular filtration barrier. However, the mechanism whereby podocytes adhere to collagen IV networks, a major component of the glomerular basement membrane, is poorly understood. The predominant collagen IV network is composed of triple helical protomers containing the alpha3alpha4alpha5 chains. The protomers connect via the trimeric noncollagenous (NC1) domains to form hexamers at the interface. Because the NC1 domains of this network can potentially support integrin-dependent cell adhesion, it was determined whether individual NC1 monomers or alpha3alpha4alpha5 hexamers support podocyte adhesion. It was found that, although human podocytes did not adhere to NC1 domains proper, they did adhere via integrin alphavbeta3 to a KRGDS motif located adjacent to alpha3NC1 domains. Because the KRGDS motif is a site of phosphorylation, its interactions with integrin alphavbeta3 may play a critical role in cell signaling in physiologic and pathologic states.

Goodpasture Antigen-binding Protein and Its Spliced Variant, Ceramide Transfer Protein, Have Different Functions in the Modulation of Apoptosis during Zebrafish Development

Human Goodpasture antigen-binding protein (GPBP) is an atypical protein kinase that phosphorylates the Goodpasture auto-antigen, the α3 chain of collagen IV. The COL4A3BP gene is alternatively spliced producing two protein isoforms: GPBP and GPBPΔ26. The latter lacks a serine-rich domain composed of 26 amino acid residues. Both isoforms also function as ceramide transfer proteins (CERT). Here, we explored the function of Gpbp and GpbpΔ26/CERT during embryogenesis in zebrafish. We cloned both splice variants of the zebrafish gene and found that they are differentially expressed during development. We used antisense oligonucleotide-mediated loss-of-function and synthetic mRNA-based gain-of-function approaches. Our results show that the loss-of-function phenotype is linked to cell death, evident primarily in the muscle of the somites, extensive loss of myelinated tracks, and brain edema. These results indicate that disruption of the nonvesicular ceramide transport is detrimental to normal embryonic development of somites and brain because of increased apoptosis. Moreover, this phenotype is mediated by Gpbp but not GpbpΔ26/CERT, suggesting that Gpbp is an important factor for normal skeletal muscle and brain development.

Goodpasture Antigen-binding Protein Is a Soluble Exportable Protein That Interacts with Type IV Collagen IDENTIFICATION OF NOVEL MEMBRANE-BOUND ISOFORMS

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.

A human-specific TNF-responsive promoter for Goodpasture antigen-binding protein

The Goodpasture antigen‐binding protein, GPBP, is a serine/threonine kinase whose relative expression increases in autoimmune processes. Tumor necrosis factor (TNF) is a pro‐inflammatory cytokine implicated in autoimmune pathogenesis. Here we show that COL4A3BP, the gene encoding GPBP, maps head‐to‐head with POLK, the gene encoding for DNA polymerase kappa (pol κ), and shares with it a 140‐bp promoter containing a Sp1 site, a TATA‐like element, and a nuclear factor kappa B (NFκB)‐like site. These three elements cooperate in the assembly of a bidirectional transcription complex containing abundant Sp1 and little NFκB that is more efficient in the POLK direction. Tumour necrosis factor cell induction is associated with Sp1 release, NFκB recruitment and assembly of a complex comparatively more efficient in the COL4A3BP direction. This is accomplished by competitive binding of Sp1 and NFκB to a DNA element encompassing a NFκB‐like site that is pivotal for the 140‐bp promoter to function. Consistently, a murine homologous DNA region, which contains the Sp1 site and the TATA‐like element but is devoid of the NFκB‐like site, does not show transcriptional activity in transient gene expression assays. Our findings identify a human‐specific TNF‐responsive transcriptional unit that locates GPBP in the signalling cascade of TNF and substantiates previous observations, which independently related TNF and GPBP with human autoimmunity.

Human Biliverdin Reductase Suppresses Goodpasture Antigen-binding Protein (GPBP) Kinase Activity THE REDUCTASE REGULATES TUMOR NECROSIS FACTOR-α-NF-κB-DEPENDENT GPBP EXPRESSION

The Ser/Thr/Tyr kinase activity of human biliverdin reductase (hBVR) and the expression of Goodpasture antigen-binding protein (GPBP), a nonconventional Ser/Thr kinase for the type IV collagen of basement membrane, are regulated by tumor necrosis factor (TNF-α). The pro-inflammatory cytokine stimulates kinase activity of hBVR and activates NF-κB, a transcriptional regulator of GPBP mRNA. Increased GPBP activity is associated with several autoimmune conditions, including Goodpasture syndrome. Here we show that in HEK293A cells hBVR binds to GPBP and down-regulates its TNF-α-stimulated kinase activity; this was not due to a decrease in GPBP expression. Findings with small interfering RNA to hBVR and to the p65 regulatory subunit of NF-κB show the hBVR role in the initial stimulation of GPBP expression by TNF-α-activated NF-κB; hBVR was not a factor in mediating GPBP mRNA stability. The interacting domain was mapped to the 281CX10C motif in the C-terminal 24 residues of hBVR. A 7-residue peptide, KKRILHC281, corresponding to the core of the consensus D(δ)-Box motif in the interacting domain, was as effective as the intact 296-residue hBVR polypeptide in inhibiting GPBP kinase activity. GPBP neither regulated hBVR expression nor TNF-α dependent NF-κB expression. Collectively, our data reveal that hBVR is a regulator of the TNF-α-GPBP-collagen type IV signaling cascade and uncover a novel biological interaction that may be of relevance in autoimmune pathogenesis.

Human biliverdin reductase suppresses goodpasture antigen binding protein (GPBP) kinase activity: the reductase regulates TNF-α- NF-κB-dependent GPBP expression

The Ser/Thr/Tyr kinase activity of human biliverdin reductase (hBVR) and the expression of Goodpasture antigen-binding protein (GPBP), a nonconventional Ser/Thr kinase for the type IV collagen of basement membrane, are regulated by tumor necrosis factor (TNF-α). The pro-inflammatory cytokine stimulates kinase activity of hBVR and activates NF-κB, a transcriptional regulator of GPBP mRNA. Increased GPBP activity is associated with several autoimmune conditions, including Goodpasture syndrome. Here we show that in HEK293A cells hBVR binds to GPBP and down-regulates its TNF-α-stimulated kinase activity; this was not due to a decrease in GPBP expression. Findings with small interfering RNA to hBVR and to the p65 regulatory subunit of NF-κB show the hBVR role in the initial stimulation of GPBP expression by TNF-α-activated NF-κB; hBVR was not a factor in mediating GPBP mRNA stability. The interacting domain was mapped to the 281CX10C motif in the C-terminal 24 residues of hBVR. A 7-residue peptide, KKRILHC281, corresponding to the core of the consensus D(δ)-Box motif in the interacting domain, was as effective as the intact 296-residue hBVR polypeptide in inhibiting GPBP kinase activity. GPBP neither regulated hBVR expression nor TNF-α dependent NF-κB expression. Collectively, our data reveal that hBVR is a regulator of the TNF-α-GPBP-collagen type IV signaling cascade and uncover a novel biological interaction that may be of relevance in autoimmune pathogenesis.

The expression of the Goodpasture antigen-binding protein (ceramide transporter) in adult rat brain

The Goodpasture antigen-binding protein (GPBP) plays a critical role in brain development. Knockdown of GPBP leads to loss of myelinated tracts in the central nervous system and to extensive apoptosis in the brain during early embryogenesis. GPBP was initially identified as a protein associated with the autoantigen in Goodpasture autoimmune syndrome, where it was shown to be a kinase that regulates type IV collagen organization. GPBP isoforms bind and transport ceramide from the endoplasmic reticulum to the Golgi apparatus and are therefore also known as ceramide transporters (CERT). Ceramide dysregulation is involved in autoimmunity and neurodegenerative disorders. In order to analyze the possible role of GPBP in neuroinflammation and neurodegeneration we studied the basal GPBP expression in normal rat brain. High levels of immunoreactivity were detected in neurons of the cerebral cortex, hippocampal formation, the basal ganglia, the olfactory bulb and nuclei of the thalamus, the hypothalamus and the septal area. Lower expression levels of GPBP were observed widely throughout the brain, suggesting that GPBP plays an important role in central nervous system neuron function.

Precise mapping of the Goodpasture epitope(s) using phage display, site-directed mutagenesis, and surface plasmon resonance

Goodpasture disease is an autoimmune disorder mediated by circulating autoantibodies against the noncollagenous-1 (NC1) domain of the α3 chain of type IV collagen (α3(IV)NC1). The structure of Goodpasture epitope(s) has been previously mapped into two main binding regions (E(A) and E(B)) of the α3(IV)NC1 domain using a residue mutation approach on the highly related α1(IV)NC1 domain. Here we combined phage display and surface plasmon resonance technology to more precisely localize the pathogenic binding sites. Peptides mimicking the Goodpasture epitope(s) were used to identify residues involved in autoantibody binding and found involvement of eight residues previously unrecognized within and outside of the E(A) or E(B) regions. Residue involvement in pathogenic reactivity was confirmed by site-directed mutagenesis on a more divergent α2(IV)NC1 molecule. From a mutant (M1) of the α2(IV)NC1 molecule, harboring residues previously identified as belonging to the Goodpasture epitope, additional chimeras were generated on the bases of phage display findings. All these mutants were shown to display higher reactivity with circulating Goodpasture autoantibodies than the M1 mutant. Thus, our results more precisely define Goodpasture epitope determinants and open new avenues to delineate comprehensive autoantibody-blocking agents for therapeutics.