Human Protein Microarray Research Articles

Diagnosis of Parkinson's disease based on disease-specific autoantibody profiles in human sera.

Parkinson's disease (PD), hallmarked by a variety of motor disorders and neurological decline, is the second most common neurodegenerative disease worldwide. Currently, no diagnostic test exists to identify sufferers, and physicians must rely on a combination of subjective physical and neurological assessments to make a diagnosis. The discovery of definitive blood-borne biomarkers would be a major step towards early and reliable diagnosis. Despite attention devoted to this search, such biomarkers have remained elusive. In the present study, we used human protein microarrays to reveal serum autoantibodies that are differentially expressed among PD and control subjects. The diagnostic significance of each of these autoantibodies was evaluated, resulting in the selection of 10 autoantibody biomarkers that can effectively differentiate PD sera from control sera with a sensitivity of 93.1% and specificity of 100%. PD sera were also distinguishable from sera obtained from Alzheimer's disease, breast cancer, and multiple sclerosis patients with accuracies of 86.0%, 96.6%, and 100%, respectively. Results demonstrate that serum autoantibodies can be used as highly specific and accurate biomarkers for PD diagnosis throughout the course of the disease.

Regulation of CK2 by phosphorylation and O-GlcNAcylation revealed by semisynthesis

Protein Ser/Thr kinase CK2 (casein kinase II) is involved in a myriad of cellular processes including cell growth and proliferation by phosphorylating hundreds of substrates, yet the regulation process of CK2 function is poorly understood. Here we report that the CK2 catalytic subunit CK2α is modified by O-GlcNAc on Ser347, proximal to a cyclin-dependent kinase phosphorylation site (Thr344) on the same protein. We use protein semisynthesis to show that Thr344 phosphorylation increases CK2α cellular stability via Pin1 interaction whereas Ser347 glycosylation appears to be antagonistic to Thr344 phosphorylation and permissive to proteasomal degradation. By performing kinase assays with the site-specifically modified phospho- and glyco-modified CK2α in combination with CK2β and Pin1 binding partners on human protein microarrays, we show that CK2 kinase substrate selectivity is modulated by these specific posttranslational modifications. This study suggests how a promiscuous protein kinase can be regulated at multiple levels to achieve particular biological outputs.

Anti-Enteric Neuronal Antibodies and the Irritable Bowel Syndrome

Background/AimsFunctional gastrointestinal disorders are those in which no abnormal metabolic or physical processes, which can account for the symptoms, can be identified. The irritable bowel syndrome (IBS) is a significant functional disorder, which affects 10-20 percent of the population worldwide. Predominant symptoms of IBS are abnormal defecation associated with abdominal pain, both of which may be exacerbated by psychogenic stress. Our study was designed to test a hypothesis that symptoms in a subset of patients with a diagnosis of IBS are associated with an autoimmune degenerative neuropathy in the enteric nervous system.MethodsSerum was collected from Rome II-IBS patients and controls at the University of North Carolina Functional Gastrointestinal Diseases Center. Assay procedures were immunohistochemical localization of antibody binding to enteric neurons and human protein microarray assay for antigens recognized by antibodies in the sera.ResultsEighty-seven percent of IBS sera and 59% of control sera contained anti-enteric neuronal antibodies. Antibody immunostaining was seen in the nucleus and cytoplasm of neurons in the enteric nervous system. Protein microarray analysis detected antibody reactivity for autoantigens in serum with anti-enteric neuronal antibodies and no reactivity for the same autoantigens in samples not containing anti-enteric neuronal antibodies in our immunostaining assay. Antibodies in sera from IBS patients recognized only 3 antigens out of an 8,000 immunoprotein array. The 3 antigens were: (1) a nondescript ribonucleoprotein (RNP-complex); (2) small nuclear ribonuclear polypeptide A; and (3) Ro-5,200 kDa.ConclusionsResults of the present study suggest that symptoms in a subset of IBS patients might be a reflection of enteric neuronal damage or loss, caused by circulating anti-enteric autoimmune antibodies.

Novel systemic lupus erythematosus autoantigens identified by human protein microarray technology

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease affecting many organs. Many autoantibodies have been associated with the disease, but either in low specificity or low sensitivity of detection. In an aim to screen for better autoantibodies, we profiled the autoantibody repertoire in sera from 30 SLE patients versus 30 healthy controls using a protein microarray containing 5011 non-redundant human proteins, and identified four candidates. We then selected CLIC2 for further verification by ELISA in an extended cohort including 110 SLE, 121 non-AD, 118 RA, 117 SSc, and 105 pSS patients. The positive rate of anti-CLIC2 was 28.18% in SLE patients, significantly higher than those in non-AD, RA, and SSc patients. The presence of anti-CLIC2 in SLE had positive correlation with disease activity in terms of SLEDAI score and several indexes (p<0.05).

Conserved Herpesvirus Kinases Target the DNA Damage Response Pathway and TIP60 Histone Acetyltransferase to Promote Virus Replication

Herpesviruses, which are major human pathogens, establish life-long persistent infections. Although the α, β, and γ herpesviruses infect different tissues and cause distinct diseases, they each encode a conserved serine/threonine kinase that is critical for virus replication and spread. The extent of substrate conservation and the key common cell-signaling pathways targeted by these kinases are unknown. Using a human protein microarray high-throughput approach, we identify shared substrates of the conserved kinases from herpes simplex virus, human cytomegalovirus, Epstein-Barr virus (EBV), and Kaposi's sarcoma-associated herpesvirus. DNA damage response (DDR) proteins were statistically enriched, and the histone acetyltransferase TIP60, an upstream regulator of the DDR pathway, was required for efficient herpesvirus replication. During EBV replication, TIP60 activation by the BGLF4 kinase triggers EBV-induced DDR and also mediates induction of viral lytic gene expression. Identification of key cellular targets of the conserved herpesvirus kinases will facilitate the development of broadly effective antiviral strategies.

Interactions of Pathological Hallmark Proteins: TUBULIN POLYMERIZATION PROMOTING PROTEIN/p25, β-AMYLOID, AND α-SYNUCLEIN

The disordered tubulin polymerization promoting protein (TPPP/p25) was found to be co-enriched in neuronal and glial inclusions with α-synuclein in Parkinson disease and multiple system atrophy, respectively; however, co-occurrence of α-synuclein with β-amyloid (Aβ) in human brain inclusions has been recently reported, suggesting the existence of mixed type pathologies that could result in obstacles in the correct diagnosis and treatment. Here we identified TPPP/p25 as an interacting partner of the soluble Aβ oligomers as major risk factors for Alzheimer disease using ProtoArray human protein microarray. The interactions of oligomeric Aβ with proteins involved in the etiology of neurological disorders were characterized by ELISA, surface plasmon resonance, pelleting experiments, and tubulin polymerization assay. We showed that the Aβ(42) tightly bound to TPPP/p25 (K(d) = 85 nm) and caused aberrant protein aggregation by inhibiting the physiologically relevant TPPP/p25-derived microtubule assembly. The pair-wise interactions of Aβ(42), α-synuclein, and tubulin were found to be relatively weak; however, these three components formed soluble ternary complex exclusively in the absence of TPPP/p25. The aggregation-facilitating activity of TPPP/p25 and its interaction with Aβ was monitored by electron microscopy with purified proteins by pelleting experiments with cell-free extracts as well as by confocal microscopy with CHO cells expressing TPPP/p25 or amyloid. The finding that the interaction of TPPP/p25 with Aβ can produce pathological-like aggregates is tightly coupled with unusual pathology of the Alzheimer disease revealed previously; that is, partial co-localization of Aβ and TPPP/p25 in the case of diffuse Lewy body disease with Alzheimer disease.

Diagnosis of Alzheimer's Disease Based on Disease-Specific Autoantibody Profiles in Human Sera

After decades of Alzheimer's disease (AD) research, the development of a definitive diagnostic test for this disease has remained elusive. The discovery of blood-borne biomarkers yielding an accurate and relatively non-invasive test has been a primary goal. Using human protein microarrays to characterize the differential expression of serum autoantibodies in AD and non-demented control (NDC) groups, we identified potential diagnostic biomarkers for AD. The differential significance of each biomarker was evaluated, resulting in the selection of only 10 autoantibody biomarkers that can effectively differentiate AD sera from NDC sera with a sensitivity of 96.0% and specificity of 92.5%. AD sera were also distinguishable from sera obtained from patients with Parkinson's disease and breast cancer with accuracies of 86% and 92%, respectively. Results demonstrate that serum autoantibodies can be used effectively as highly-specific and accurate biomarkers to diagnose AD throughout the course of the disease.

Protein Microarrays Identify Elevated Allogeneic Antibodies In Association with Extensive Chronic Graft Versus Host Disease

AbstractAbstract 2344 Introduction:Allogeneic hematopoietic cell transplantation (HCT) can cure hematologic malignancies through beneficial graft versus leukemia (GVL) allo-immune responses, but is limited by graft versus host disease (GVHD). Studies have shown allogeneic antibodies (allo-Ab) develop against minor histocompatibility antigens encoded on the Y-chromosome after sex-mismatch HCT in association with chronic GVHD (cGVHD) and persistent disease remission. Recombinant human protein microarrays facilitate multiplex antibody quantification and have successfully identified GVL antigens in single patient studies. This study applies protein microarray technology to compare antibody development in 43 leukemia patients in relation to allogeneic HCT outcomes. We hypothesized allo-Ab quantification may identify patients with cGVHD and identify unique GVL antigens in acute myeloid or lymphoblastic leukemia. Method:We enrolled a consecutive series of acute leukemia patients undergoing HLA-identical myeloablative allogeneic HCT and studied the 20 AML and 23 ALL patients who survived at least one year after transplantation. Patients received FK506 and methotrexate immune prophylaxis. Only 5 of 43 grafts were bone marrow and 33% of donors were unrelated. Extensive cGVHD developed in 24 of 43 (56%) patients. Plasma samples collected pre-HCT, one year post-transplant, and from 45 healthy donors were analyzed for antibodies using v4.1 ProtoArrays (Invitrogen, Carlsbad, CA). These are nitrocellulose coated glass slides printed in duplicate with 8350 unique human proteins epitope-tagged with GST. Plasma diluted 1:500 was incubated 1.5 hours and bound antibodies were detected using fluorochrome conjugated anti-human IgG. Slide processing and fluorochrome detection were normalized by comparison to known microarray spotted IgG concentrations. Overall, mean fluorescence intensity (MFI) of duplicate spots showed excellent agreement (R2=0.95). Each antibody response was normalized for target protein concentration using anti-GST measurements. Antibody responses were reported as Z scores (number of standard deviations above each sample's average MFI). For each sample, the sum of Z scores>2 (SZ2) was analyzed in relation to clinical outcome. Results:The SZ2 measured one year post-HCT ranged from 279 to 6652 and did not correlate with total IgG (R2=0.26). Univariate analysis of transplant risk factors and clinical outcomes showed SZ2 associated with extensive chronic GVHD. Using a SZ2 cutoff of 3700, the relative risk of developing extensive cGVHD was 1.9 (95%CI 1.14–3.07) with a positive predictive value of 84.2%. These allo-Ab (SZ2>3700) developed in extensive cGVHD patients despite elevated corticosteroid therapy (p<0.001). Further, a set of 25 proteins were recognized by allo-Ab in more than 50% of cGVHD patients including fibroblast growth factor 12. Strong allo-Ab responses developed against FGF12 in 16/24 (66%) of extensive cGVHD patients (Z scores ranging 3–22, median of 8).With a median post-HCT follow-up of 2.7 years, only 6 patients have relapsed and SZ2 did not associate. In order to identify AML and ALL specific GVL antigens, we screened for allo-Ab targets exclusively recognized in either AML or ALL patients but not detected in 45 normal donors. Twelve AML and 20 ALL candidate GVL targets were identified in at least 6 of 20 (30%) patients including Myosin Light Chain Kinase 2 (MYLK2) in AML patients. These candidate GVL antigens are currently being validated using banked samples collected after allo-HCT from patients with a variety of malignancies. Conclusion:Protein microarray technology and normalization methods enable quantitative antibody measurements across complex patient populations. Here we show elevated allogeneic antibodies measured by protein microarray one year after transplant associated with extensive cGVHD development. We conclude protein microarrays can identify GVHD antibody signatures and that total allo-Ab quantification can be monitored in real-time for cGVHD development aiding immune suppression management. Disclosures:Miklos: Novartis: Honoraria, Research Funding.

Identification of a novel autoantigen in inflammatory bowel disease by protein microarray

Patients with inflammatory bowel disease (IBD) display immunoreactivity to self-antigens and microbial antigens. We used a protein microarray approach to identify novel autoantigens in IBD. ProtoArray Human Protein Microarray v4.0 containing 8268 human proteins from Invitrogen (La Jolla, CA) was used. Twenty-five IBD patients and five healthy controls were screened for candidate autoantigens. For 256 antigens, IBD patients had a higher seroreactivity than controls. Twenty antigens were selected for further evaluation in a larger cohort (60 ulcerative colitis [UC] patients, 60 Crohn's disease [CD] patients, 60 healthy controls, and 60 gastrointestinal-diseased controls) by means of a customized protein microarray. Out of these 20 antigens, one antigen, family with sequence similarity 84 member A (FAM84A), was identified as a target antigen in IBD. Antibodies to FAM84A were significantly more prevalent in IBD patients (19%) than in gastrointestinal-diseased controls (1.7%) (P = 0.0008) and healthy controls (5%) (P = 0.01). Anti-FAM84A antibodies were found in 26.6% of UC patients and in 11.7% of CD patients. FAM84A was confirmed as target antigen in IBD by means of Western blotting in a large independent cohort (100 UC patients, 106 CD patients, 102 healthy controls, and 100 gastrointestinal-diseased controls). Antibodies to FAM84A were significantly more prevalent in IBD patients (20%) than in gastrointestinal-diseased controls (5%) (P = 0.0004) and healthy controls (0%) (P < 0.0001). Anti-FAM84A antibodies were found in 18% of UC patients and in 22% of CD patients. We identified FAM84A as a novel autoantigen in IBD. (Inflamm Bowel Dis 2011;).

Identification of Substrates of SMURF1 Ubiquitin Ligase Activity Utilizing Protein Microarrays

The ubiquitin proteasome pathway (UPP) has been implicated in a number of pathogenic diseases: cancer, inflammation, metabolic disorders, and viral infection. The human genome contains well over 500 genes encoding proteins involved in the UPP. Ubiquitin ligases (E3s) comprise the largest subset of these genes, and together with an E2 partner, provide the substrate selectivity required for regulating cellular proteins through the covalent attachment of ubiquitin. Many ligases that have been identified in critical cellular pathways have no known substrates. Even those E3s with known substrates may have a yet unidentified role in the pathways on which they lie and as such may have additional substrates. It is critical to identify these substrates for discovery of selective small molecule inhibitors aimed at therapeutic intervention. Other methods, such as mass spectrometry, have been utilized for identifying ligase substrates, but these are labor-intensive and require a significant investment. In this study, we utilized protein microarrays for the identification of substrates of the HECT domain E3, Smurf1. Smurf1 is a critical regulator of TGF-beta and bone morphogenic protein signaling, and has been demonstrated to play a role in regulating cell polarity through the degradation of RhoA. We set out to identify novel Smurf1 substrates involved in the regulation of the aforementioned pathways. Proof-of-principle experiments with known Smurf1 substrates demonstrated efficient ubiquitination thereby validating this approach. Assaying a human protein microarray for ubiquitination with Smurf1 and the partner E2 ubiquitin ligase Ubch5 or Ubch7 identified 89 potential substrates of the Smurf1 E3 activity, which spanned a number of different biological pathways. Substrates identified utilizing protein microarray technology have been validated in vitro. Here we demonstrate the utility of this approach for identifying substrates of particular E2/E3 complexes.

Protein microarray analysis identifies cyclic nucleotide phosphodiesterase as an interactor of Nogo-A

Nogo-A, a neurite outgrowth inhibitor, is expressed exclusively on oligodendrocytes and neurons in the CNS. The central domain of Amino-Nogo spanning amino acids 567-748 in the human Nogo-A designated NIG, mediates persistent inhibition of axonal outgrowth and induces growth cone collapse by signaling through an as yet unidentified NIG receptor. We identified 82 NIG-interacting proteins by screening a high-density human protein microarray composed of 5000 proteins with a recombinant NIG protein as a probe. Following an intensive database search, we selected 12 neuron/oligodendrocyte-associated NIG interactors. Among them, we verified the molecular interaction of NIG with 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNP), a cell type-specific marker of oligodendrocytes, by immunoprecipitation and cell imaging analysis. Although CNP located chiefly in the cytoplasm of oligodendrocytes might not serve as a cell-surface NIG receptor, it could act as a conformational stabilizer for the intrinsically unstructured large segment of Amino-Nogo.

Applications of Functional Protein Microarrays

Comprehensive proteome-scale protein microarrays can be used to simultaneously screen up to several thousand proteins for drug binding, molecular interactions, or enzymatic activity. The ProtoArray ® Human Protein Microarray v4.0 is comprised of over 8,000 human proteins spotted in duplicate on glass slides. The arrayed proteins are expressed as full-length, N-terminal glutathione S-transferase (GST) fusion proteins in a baculovirus system. The proteins are purified under non-denaturing conditions to maximize proper folding and functionality. Examples will be given about how arrays of human proteins have been used to define protein interaction pathways, screen for enzyme substrates, measure enzyme inhibition, and identify protein targets of drugs. In addition, we have demonstrated the utility of these arrays for determining the specificity of antibodies, thus providing a new tool for the development and characterization of therapeutic and diagnostic antibodies. Finally, we will show how these arrays have been used to provide a rapid and sensitive profiling platform to investigate the circulating antibody profile in several disease states.

Allogeneic Antibodies Identify GVL Targets CHAF1b and NuSAP1 in AML Patients.

Allogeneic Hematopoietic Cell Transplantation (HCT) can cure hematologic malignancies through beneficial graft-v-leukemia (GVL) allo-immune responses, but is limited by graft-versus-host disease (GVHD). Previous studies demonstrate allogeneic antibodies (Ab) develop against minor histocompatibility antigens (mHA) encoded on the Y-chromosome (H-Y antigens) after sex-mismatch HCT in association with chronic GVHD and persistent disease remission. We hypothesize that novel mHA can be serologically identified as targets of allo-Ab responses that develop post-transplant. For this study, plasma was collected from five AML patients one year post-transplant, pre-transplant, and from their donors. The technology used to identify allo-Ab responses was human protein microarrays. ProtoArray™ (Invitrogen) displays 5,000 full-length human proteins with N-terminal GST epitopes expressed in baculovirus and affinity purified under native conditions maintaining their cellular enzymatic activities/native conformations. Technical replicates of 10 plasma measured at 1:50, 1:150, 1:500, 1:1500 and 1:4500 provided correlation coefficients of R2=0.94–0.97 confirming technical feasibility. In order to identify targets of allo-Ab, pre-transplant fluorescent signal intensities were subtracted from their one-year plasma results and also screened for the absence in donor sera for all 5000 antigens. While Ab responses were unchanged for 4600 (92%) antigens, new allo-Ab responses targeted 60–75 antigens with fluorescent differences ranging 0.5 to 3 logs. Over 90% of allo-Ab targets have known non-synonymous SNP which when disparate in donor and recipient may elicit alloimmunity. From the index patient, Nucleolar and Spindle Associated Protein 1 (NuSAP1) and Chromatin Assembly Factor 1b (CHAF1b) were two predominant proteins recognized after HCT. These allo-Abs were absent pre and 2 months post-HCT, developed in association with cGVHD at 12 months, and persisted through 18 months. On comparing all the five AML patients using ProtoArrays, we identified two patients of five developed all-ab responses to CHAF1b and NuSAP1. CHAF1b and NuSAP1 were expressed in insect cells and in E.coli, and were validated using ELISA and Western Blotting. We hypothesize alloimmunity in the index patient resulted from disparity between the donor and the recipient. We sequenced all exons for CHAF1b and NuSAP1 but failed to identify disparate non-synonymous coding SNPs between the donor and the index patient. A second hypothesis for immunogeneicity assumes CHAF1b and NuSAP1 are aberrantly expressed as tumor antigens stimulating donor allo immunity after transplantation. We determined if the antibody correlated with disease type. Eight-seven allo-transplant patients after one year post-transplant were screened by ELISA for antibody responses against CHAF1b and NuSAP1. Antibodies against NuSAP1 and CHAF1b are significantly recognized in patients with acute myelogenous leukemia (n=27) as compared to ALL, CLL, CML, MCL, MM and NHL, with a significance of p=0.00028 for CHAF1 and p=0.0011 for NuSAP1 using Wilcox test. Analysis of published AML expression profiles demonstrate CHAF1b and NuSAP1 were mainly present in the bone marrow CD34+ cells and leukemic cell lines and B lymphoblasts as compared to other tissues/cells. We sorted different cell populations and confirmed by RT-PCR results that CHAF1b and NuSAP1 were highly expressed in bone marrow CD34+Thy1+ cells suggesting antibodies specifically target bone marrow stem cells after HCT.

A Consolidated Approach to Analyzing Data from High-Throughput Protein Microarrays with an Application to Immune Response Profiling in Humans

DNA microarrays are a well-known and established technology in biological and pharmaceutical research providing a wealth of information essential for understanding biological processes and aiding drug development. Protein microarrays are quickly emerging as a follow-up technology, which will also begin to experience rapid growth as the challenges in protein to spot methodologies are overcome. Like DNA microarrays, their protein counterparts produce large amounts of data that must be suitably analyzed in order to yield meaningful information that should eventually lead to novel drug targets and biomarkers. Although the statistical management of DNA microarray data has been well described, there is no available report that offers a successful consolidated approach to the analysis of high-throughput protein microarray data. We describe the novel application of a statistical methodology to analyze the data from an immune response profiling assay using human protein microarray with over 5000 proteins on each chip.

A Critical Role for Cortactin Phosphorylation by Abl-Family Kinases in PDGF-Induced Dorsal-Wave Formation

Proper regulation of cell morphogenesis and migration by adhesion and growth-factor receptors requires Abl-family tyrosine kinases [1-3]. Several substrates of Abl-family kinase have been identified, but they are unlikely to mediate all of the downstream actions of these kinases on cytoskeletal structure. We used a human protein microarray to identify the actin-regulatory protein cortactin as a novel substrate of the Abl and Abl-related gene (Arg) nonreceptor tyrosine kinases. Cortactin stimulates cell motility [4-6], and its upregulation in several cancers correlates with poor prognosis [7]. Even though cortactin can be tyrosine phosphorylated by Src-family kinases in vitro [8], we show that Abl and Arg are more adept at binding and phosphorylating cortactin. Importantly, we demonstrate that platelet-derived growth-factor (PDGF)-induced cortactin phosphorylation on three tyrosine residues requires Abl or Arg. Cortactin triggers F-actin-dependent dorsal waves in fibroblasts after PDGF treatment and thus results in actin reorganization and lamellipodial protrusion [9]. We provide evidence that Abl/Arg-mediated phosphorylation of cortactin is required for this PDGF-induced dorsal-wave response. Our results reveal that Abl-family kinases target cortactin as an effector of cytoskeletal rearrangements in response to PDGF.

Distribution of the AQP4 Water Channel in Normal Human Tissues: Protein and Tissue Microarrays Reveal Expression in Several New Anatomical Locations, including the Prostate Gland and Seminal Vesicles

Aquaporins facilitate osmotically driven water movement across cell membranes. Aquaporin 4 (AQP4) is a major water channel in the central nervous system where it participates in cerebral water balance. AQP4 is also present in basolateral membranes of lower respiratory tract airway and renal collecting duct epithelial cells, gastric parietal cells and skeletal muscle cells. However, the distribution of AQP4 in many other tissues is still unknown. The aim of this study was to determine the expression and relative abundance of AQP4 in human Tissue MicroArrays (TMAs) and human protein microarrays by immunohistochemistry and chemiluminescence. In the central nervous system AQP4 was abundantly expressed in the cerebral cortex, cerebellar cortex (purkinje/granular layer), ependymal cell layer, hippocampus and spinal cord. Lower levels were detected in choroid plexus, white matter and meninges. In the musculoskeletal system AQP4 was highly expressed in the sarcolemma of skeletal muscle from the chest and neck. In the male genital system AQP4 was moderately expressed in seminiferous tubules, seminal vesicles, prostate and epidiymis. In the respiratory system AQP4 was moderately expressed in lung and bronchus. AQP expression was abundant in the kidney. In the gastrointestinal system AQP4 was moderately present in basolateral membranes of parietal cells at the base of gastric glands. AQP4 was also detected in salivary glands, adrenals, anterior pituitary, prostate and seminal vesicles. Human protein microarrays verified the TMA data. Our findings suggest that AQP4 is expressed more widely than previously thought in human organs and may be involved in prostatic and seminal fluid formation.