O-Glycomic and Proteomic Signatures of Spontaneous and Butyrate-Stimulated Colorectal Cancer Cell Line Differentiation.

Gut microbiota of the gastrointestinal tract provide health benefits to the human host via bacterial metabolites. Bacterial butyrate has beneficial effects on intestinal homeostasis and is the preferred energy source of intestinal epithelial cells, capable of inducing differentiation. It was previously observed that changes in the expression of specific proteins as well as protein glycosylation occur with differentiation. In this study, specific mucin O-glycans were identified that mark butyrate-induced epithelial differentiation of the intestinal cell line CaCo-2 (Cancer Coli-2), by applying porous graphitized carbon nano-liquid chromatography with electrospray ionization tandem mass spectrometry. Moreover, a quantitative proteomic approach was used to decipher changes in the cell proteome. It was found that the fully differentiated butyrate-stimulated cells are characterized by a higher expression of sialylated O-glycan structures, whereas fucosylation is downregulated with differentiation. By performing an integrative approach, we generated hypotheses about the origin of the observed O-glycome changes. These insights pave the way for future endeavors to study the dynamic O-glycosylation patterns in the gut, either produced via cellular biosynthesis or through the action of bacterial glycosidases as well as the functional role of these patterns in homeostasis and dysbiosis at the gut-microbiota interface.

Retina-arrestin specific CD8+ T cells are not implicated in HLA-A29-positive birdshot chorioretinitis.

BACKGROUND: HLA-A29-positive birdshot chorioretinitis (BCR) is an inflammatory eye disorder that is generally assumed to be caused by an autoimmune response to HLA-A29-presented peptides from retinal arrestin (SAG), yet the epitopes recognized by CD8+ T cells from patients remain to be identified. OBJECTIVES: The identification of natural ligands of SAG presented by HLA-A29. To quantify CD8+ T cells reactive to antigenic SAG peptides presented by HLA-A29 in patients and controls. METHODS: We performed mass-spectrometry based immunopeptidomics of HLA-A29 of antigen-presenting cell lines from patients engineered to express SAG. MHC-I Dextramer technology was utilised to determine expansion of antigen-specific CD8+ T cells reactive to SAG peptides in complex with HLA-A29 in a cohort of BCR patients, HLA-A29-positive controls, and HLA-A29-negative controls. RESULTS: We report on the naturally presented antigenic SAG peptides identified by sequencing the HLA-A29 immunopeptidome of antigen-presenting cells of patients. We show that the N-terminally extended SAG peptide precursors can be trimmed in vitro by the antigen-processing aminopeptidases ERAP1 and ERAP2. Unexpectedly, no enhanced antigen engagement by CD8+ T cells upon stimulation with SAG peptides was observed in patients or HLA-A29-positive controls. Multiplexed HLA-A29-peptide dextramer profiling of a case-control cohort revealed that CD8+ T cells specific for these SAG peptides were neither detectable in peripheral blood nor in eye biopsies of patients. CONCLUSIONS: Collectively, these findings demonstrate that SAG is not a CD8+ T cell autoantigen and sharply contrast the paradigm in the pathogenesis of BCR. Therefore, the mechanism by which HLA-A29 is associated with BCR does not involve SAG.

WWP2 confers risk to osteoarthritis by affecting cartilage matrix deposition via hypoxia associated genes.

OBJECTIVE: To explore the co-expression network of the osteoarthritis (OA) risk gene WWP2 in articular cartilage and study cartilage characteristics when mimicking the effect of OA risk allele rs1052429-A on WWP2 expression in a human 3D in vitro model of cartilage. METHOD: Co-expression behavior of WWP2 with genes expressed in lesioned OA articular cartilage (N = 35 samples) was explored. By applying lentiviral particle mediated WWP2 upregulation in 3D in vitro pellet cultures of human primary chondrocytes (N = 8 donors) the effects of upregulation on cartilage matrix deposition was evaluated. Finally, we transfected primary chondrocytes with miR-140 mimics to evaluate whether miR-140 and WWP2 are involved in similar pathways. RESULTS: Upon performing Spearman correlations in lesioned OA cartilage, 98 highly correlating genes (|ρ| > 0.7) were identified. Among these genes, we identified GJA1, GDF10, STC2, WDR1, and WNK4. Subsequent upregulation of WWP2 on 3D chondrocyte pellet cultures resulted in a decreased expression of COL2A1 and ACAN and an increase in EPAS1 expression. Additionally, we observed a decreased expression of GDF10, STC2, and GJA1. Proteomics analysis identified 42 proteins being differentially expressed with WWP2 upregulation, which were enriched for ubiquitin conjugating enzyme activity. Finally, upregulation of miR-140 in 2D chondrocytes resulted in significant upregulation of WWP2 and WDR1. CONCLUSIONS: Mimicking the effect of OA risk allele rs1052429-A on WWP2 expression initiates detrimental processes in the cartilage shown by a response in hypoxia associated genes EPAS1, GDF10, and GJA1 and a decrease in anabolic markers, COL2A1 and ACAN.

Carbamylation reduces the capacity of IgG for hexamerization and complement activation.

Carbamylation is a post-translational modification that can be detected on a range of proteins, including immunoglobulin (Ig)G, in several clinical conditions. Carbamylated IgG (ca-IgG) was reported to lose its capacity to trigger complement activation, but the mechanism remains unclear. Because C1q binds with high affinity to hexameric IgG, we analyzed whether carbamylation of IgG affects binding of C1q, hexamerization and complement-dependent cytotoxicity (CDC). Synovial tissues of rheumatoid arthritis (RA) patients were analyzed for the presence of ca-IgG in vivo. Synovial tissues from RA patients were analyzed for the presence of ca-IgG using mass spectrometry (MS). Monomeric or hexameric antibodies were carbamylated in vitro and quality in solution was controlled. The capacity of ca-IgG to activate complement was analyzed in enzyme-linked immunosorbent (ELISAs) and cellular CDC assays. Using MS, we identified ca-IgG to be present in the joints of RA patients. Using in vitro carbamylated antibodies, we observed that ca-IgG lost its capacity to activate complement in both solid-phase and CDC assays. Mixing ca-IgG with non-modified IgG did not result in effective inhibition of complement activation by ca-IgG. Carbamylation of both monomeric IgG and preformed hexameric IgG greatly impaired the capacity to trigger complement activation. Furthermore, upon carbamylation, the preformed hexameric IgG dissociated into monomeric IgG in solution, indicating that carbamylation influences both hexamerization and C1q binding. In conclusion, ca-IgG can be detected in vivo and has a strongly reduced capacity to activate complement which is, in part, mediated through a reduced ability to form hexamers.

Antibodies and B cells recognising citrullinated proteins display a broad cross-reactivity towards other post-translational modifications.

OBJECTIVE: Autoantibodies against antigens carrying distinct post-translational modifications (PTMs), such as citrulline, homocitrulline or acetyllysine, are hallmarks of rheumatoid arthritis (RA). The relation between these anti-modified protein antibody (AMPA)-classes is poorly understood as is the ability of different PTM-antigens to activate B-cell receptors (BCRs) directed against citrullinated proteins (CP). Insights into the nature of PTMs able to activate such B cells are pivotal to understand the 'evolution' of the autoimmune response conceivable underlying the disease. Here, we investigated the cross-reactivity of monoclonal AMPA and the ability of different types of PTM-antigens to activate CP-reactive BCRs. METHODS: BCR sequences from B cells isolated using citrullinated or acetylated antigens were used to produce monoclonal antibodies (mAb) followed by a detailed analysis of their cross-reactivity towards PTM-antigens. Ramos B-cell transfectants expressing CP-reactive IgG BCRs were generated and their activation on stimulation with PTM-antigens investigated. RESULTS: Most mAbs were highly cross-reactive towards multiple PTMs, while no reactivity was observed to the unmodified controls. B cells carrying CP-reactive BCRs showed activation on stimulation with various types of PTM-antigens. CONCLUSIONS: Our study illustrates that AMPA exhibit a high cross-reactivity towards at least two PTMs indicating that their recognition pattern is not confined to one type of modification. Furthermore, our data show that CP-reactive B cells are not only activated by citrullinated, but also by carbamylated and/or acetylated antigens. These data are vital for the understanding of the breach of B-cell tolerance against PTM-antigens and the possible contribution of these antigens to RA-pathogenesis.

Human islets and dendritic cells generate post-translationally modified islet autoantigens.

The initiation of type 1 diabetes (T1D) requires a break in peripheral tolerance. New insights into neoepitope formation indicate that post-translational modification of islet autoantigens, for example via deamidation, may be an important component of disease initiation or exacerbation. Indeed, deamidation of islet autoantigens increases their binding affinity to the T1D highest-risk human leucocyte antigen (HLA) haplotypes HLA-DR3/DQ2 and -DR4/DQ8, increasing the chance that T cells reactive to deamidated autoantigens can be activated upon T cell receptor ligation. Here we investigated human pancreatic islets and inflammatory and tolerogenic human dendritic cells (DC and tolDC) as potential sources of deamidated islet autoantigens and examined whether deamidation is altered in an inflammatory environment. Islets, DC and tolDC contained tissue transglutaminase, the key enzyme responsible for peptide deamidation, and enzyme activity increased following an inflammatory insult. Islets treated with inflammatory cytokines were found to contain deamidated insulin C-peptide. DC, heterozygous for the T1D highest-risk DQ2/8, pulsed with native islet autoantigens could present naturally processed deamidated neoepitopes. HLA-DQ2 or -DQ8 homozygous DC did not present deamidated islet peptides. This study identifies both human islets and DC as sources of deamidated islet autoantigens and implicates inflammatory activation of tissue transglutaminase as a potential mechanism for islet and DC deamidation.

Identification and characterisation of peptide binding motifs of six autoimmune disease-associated human leukocyte antigen-class I molecules including HLA-B*39:06.

Research on CD8 T cell-mediated inflammatory diseases requires a better understanding of target epitopes and the constraints placed upon these by major histocompatibility complex (MHC) class I binding restrictions, especially those that relate to predisposing alleles. We used linear trap quadrupole fourier transform (LTQ-FT) tandem mass spectrometry to identify naturally processed and presented peptides eluted from the MHC-negative myeloid leukaemia cell line K562 transfected with specific MHC class I genes. We provide information on the peptidome of HLA-B*39:06, which is associated with the autoimmune disease type 1 diabetes, and extend the analysis to include a further five human leukocyte antigen (HLA) alleles (HLA-A*02:01/-A*11:01/-A*24:02/-B*18:01/-B*38:01) studied under identical experimental conditions. We identified a total of 3095 individual peptides with a mascot score ≥40 (HLA-A*02:01 = 569 peptides, -A*11:01 = 904, A*24:02 = 257, -B*18:01 = 615, -B*38:01 = 453, -B*39:06 = 297). Peptides had a preferential length of nine amino acids and originated mainly from cytoplasmic or nuclear proteins. Eluted peptides revealed a strong binding motif with binding anchor positions at position 2 (P2) and the C-terminus (PΩ). Peptides eluted from HLA-A*02:01 showed a P2 preference for leucine (62% of total peptides have Leu at P2) and PΩ preference for valine (49%). Similar data are provided for HLA-A*11:01 (P2:Thr, 29%; PΩ:Lys, 49%), -A*24:02 (P2:Tyr, 78%; PΩ:Phe, 41%), -B*18:01 (P2:Glu, 77%; PΩ:Tyr, 32%), -B*38:01 (P2:His, 51%; PΩ:Leu, 45%) and -B*39:06 (P2:Arg/His, 24%; PΩ:Ala, 64%). This work thus gives an overview of the naturally processed and presented repertoire of several common and autoimmune disease-related HLA alleles, which may be useful in studying autoreactive CD8 T cell responses and the role of HLA in disease susceptibility.

Efficient identification of novel HLA-A(*)0201-presented cytotoxic T lymphocyte epitopes in the widely expressed tumor antigen PRAME by proteasome-mediated digestion analysis.

We report the efficient identification of four human histocompatibility leukocyte antigen (HLA)-A(*)0201-presented cytotoxic T lymphocyte (CTL) epitopes in the tumor-associated antigen PRAME using an improved "reverse immunology" strategy. Next to motif-based HLA-A(*)0201 binding prediction and actual binding and stability assays, analysis of in vitro proteasome-mediated digestions of polypeptides encompassing candidate epitopes was incorporated in the epitope prediction procedure. Proteasome cleavage pattern analysis, in particular determination of correct COOH-terminal cleavage of the putative epitope, allows a far more accurate and selective prediction of CTL epitopes. Only 4 of 19 high affinity HLA-A(*)0201 binding peptides (21%) were found to be efficiently generated by the proteasome in vitro. This approach avoids laborious CTL response inductions against high affinity binding peptides that are not processed and limits the number of peptides to be assayed for binding. CTL clones induced against the four identified epitopes (VLDGLDVLL, PRA(100-108); SLYSFPEPEA, PRA(142-151); ALYVDSLFFL, PRA(300-309); and SLLQHLIGL, PRA(425-433)) lysed melanoma, renal cell carcinoma, lung carcinoma, and mammary carcinoma cell lines expressing PRAME and HLA-A(*)0201. This indicates that these epitopes are expressed on cancer cells of diverse histologic origin, making them attractive targets for immunotherapy of cancer.

Pitfalls in the detection of citrullination and carbamylation.

Carbamylation and citrullination are both post-translational modifications against which (auto)antibodies can be detected in sera of rheumatoid arthritis (RA) patients. Carbamylation is the chemical modification of a lysine into a homocitrulline, whereas citrullination is an enzymatic conversion of an arginine into a citrulline. It is difficult to distinguish between the two resulting amino acids due to similarities in structure. However, differentiation between citrulline and homocitrulline is important to understand the antigens that induce antibody production and to determine which modified antigens are present in target tissues. We have observed in literature that conclusions are frequently drawn regarding the citrullination or carbamylation of proteins based on reagents that are not able to distinguish between these two modifications. Therefore, we have analyzed a wide spectrum of methods and describe here which method we consider most optimal to distinguish between citrulline and homocitrulline. We have produced several carbamylated and citrullinated proteins and investigated the specificity of (commercial) antibodies by both ELISA and western blot. Furthermore, detection methods based on chemical modifications, such as the anti-modified citrulline-"Senshu" method and also mass spectrometry were investigated for their capacity to distinguish between carbamylation and citrullination. We observed that some antibodies are able to distinguish between carbamylation and citrullination, but an overlap in reactivity is often present in the commercially available anti-citrulline antibodies. Finally, we conclude that the use of mass spectrometry is currently essential to differentiate between citrullinated and carbamylated proteins present in complex biological samples.

BCR-ABL fusion regions as a source of multiple leukemia-specific CD8+ T-cell epitopes.

For immunotherapy of residual disease in patients with Philadelphia-positive leukemias, the BCR-ABL fusion regions are attractive disease-specific T-cell targets. We analyzed these regions for the prevalence of cytotoxic T lymphocyte (CTL) epitopes by an advanced reverse immunology procedure. Seventeen novel BCR-ABL fusion peptides were identified to bind efficiently to the human lymphocyte antigen (HLA)-A68, HLA-B51, HLA-B61 or HLA-Cw4 HLA class I molecules. Comprehensive enzymatic digestion analysis showed that 10 out of the 28 HLA class I binding fusion peptides were efficiently excised after their C-terminus by the proteasome, which is an essential requirement for efficient cell surface expression. Therefore, these peptides are prime vaccine candidates. The other peptides either completely lacked C-terminal liberation or were only inefficiently excised by the proteasome, rendering them inappropriate or less suitable for inclusion in a vaccine. CTL raised against the properly processed HLA-B61 epitope AEALQRPVA from the BCR-ABL e1a2 fusion region, expressed in acute lymphoblastic leukemia (ALL), specifically recognized ALL tumor cells, proving cell surface presentation of this epitope, its applicability for immunotherapy and underlining the accuracy of our epitope identification strategy. Our study provides a reliable basis for the selection of optimal peptides to be included in immunotherapeutic BCR-ABL vaccines against leukemia.

Processing and targeting of a molluscan egg-laying peptide prohormone as revealed by mass spectrometric peptide fingerprinting and peptide sequencing.

The neuroendocrine cerebral caudodorsal cells of Lymnaea stagnalis initiate and coordinate ovulation and egg mass production and associated behaviors through the release of a complex set of peptides that are derived from the caudodorsal cell hormone-I (CDCH-I) precursor. We have previously characterized the CDCH-I peptide. In the present study, we isolated and amino acid sequenced by conventional peptide chemistry five additional peptides, epsilon-peptide, calfluxin, alpha-caudodorsal cell peptide, delta-peptide, and carboxyl-terminally located peptide, from the cerebral commissure, the neurohemal area of the caudodorsal cells. Fingerprinting by matrix-assisted laser desorption mass spectrometry of peptides in the commissure demonstrated the presence of all sequenced peptides and, in addition, could identify two other peptides derived from pro-CDCH-1, the beta 1- and beta 3-peptides. These findings together with previous immunocytochemical studies enabled us to define cleavage sites and major processing events of pro-CDCH-1. Pro-CDCH-1 is initially cleaved in the Golgi apparatus into carboxyl- and amino-terminal parts, each of which is sorted into distinct vesicle classes that traffic to different intracellular sites. As a result, in the commissure, peptides derived from the carboxyl-terminal part, including CDCH-1, are present at a many-fold higher concentration than those derived from the amino-terminal part.

Structural identification, neuronal synthesis, and role in male copulation of myomodulin-A of Lymnaea: a study involving direct peptide profiling of nervous tissue by mass spectrometry.

We used a strategy combining immunodetection, peptide chemistry, and a novel method, direct peptide fingerprinting of neurons and small pieces of nerve by using matrix-assisted laser desorption ionization mass spectrometry, to structurally identify and localize the neuropeptide myomodulin-A in the mollusc, Lymnaea stagnalis. Lymnaea myomodulin appeared to be identical to Aplysia myomodulin-A and is produced by many central neurons, including neurons located in the ventral lobe of the right cerebral ganglion that innervate the penis complex via the penis nerve. Myomodulin-A could also be characterized from the penis complex, and physiological concentrations of the peptide enhanced the relaxation rate of electrically induced contractions of the penis retractor muscle in vitro in a dose-dependent fashion.

Structure, localization and action of a novel inhibitory neuropeptide involved in the feeding of Lymnaea.

A neuropeptide that strongly inhibits the spontaneous contractions of the oesophagus in Lymnaea has been characterized as GAPRFVamide. Direct mass spectrometry of nervous tissues and immunocytochemical studies show that the peptide is synthesized by neurones in the buccal ganglia and transported to the oesophagus via the dorso-buccal nerve. In accordance with the function of the peptide, immunoreactive fibres are detected within the muscle layer of the oesophagus. Finally, mass spectrometry reveals the presence of a number of unidentified peptides in the nerves that innervate the oesophagus, which suggests that oesophageal activities may be modified by multiple peptides.

Structure elucidation of gluten-derived peptides by tandem mass spectrometry.

In recent years there have been major developments in the field of mass spectrometry (MS) that permit the analysis and characterization of peptides and proteins at the femtomolar level (1-8). This chapter deals with the use of MS for the elucidation of peptide sequences of gliadin- and/or glutenin-derived fragments. It is meant to give an overview of what modern mass spectrometric methods can mean for your research in the biomolecular field rather than an in-depth step-by-step practical protocol. Important aspects are covered to give the reader an idea of what is needed to perform, and what can be expected from, this mass spectrometric application.

Polyubiquitination and proteasomal turnover controls the anti-apoptotic activity of Bcl-B.

Anti-apoptotic Bcl-2 family members can contribute to tumorigenesis and may convey resistance to anti-cancer regimens. Therefore, they are important targets for novel therapeutics, particularly Bcl-2 homology (BH)3 mimetics. Bcl-B (BCL-2-like protein-10) is a relatively understudied member of the Bcl-2 protein family. Its physiological function is unknown, but it has been proven to have an anti-apoptotic activity and to act as a tumor promoter in mice. In human, high Bcl-B protein expression levels correlate with poor prognosis in various carcinomas and predict treatment resistance in acute myeloid leukemia. We here report that protein expression level and anti-apoptotic activity of Bcl-B are dictated by its ubiquitination. We demonstrate that Bcl-B is polyubiquitinated at steady state, in a unique loop between the BH1 and BH2 domains. Mutagenesis identified lysine (K)128 as an acceptor site for polyubiquitin chains, and K119 and K120, but not K181, as potential ubiquitination sites. Mass spectrometry confirmed K128 as a ubiquitination site and defined the polyubiquitin chains as K48-linked, which was confirmed by linkage-specific antibodies. Accordingly, Bcl-B proved to be an instable protein that is subject to ubiquitin-dependent proteasomal degradation at steady state. At equal mRNA expression, protein expression of a lysineless, nonubiquitinated Bcl-B mutant was fivefold higher than that of wild-type Bcl-B, demonstrating that ubiquitination is a key determinant for Bcl-B protein expression levels. Ubiquitination controlled the anti-apoptotic capacity of Bcl-B, in response to a variety of conventional and novel anti-cancer drugs. Certain anti-cancer drugs, known to reduce Mcl-1 protein levels, likewise downregulated Bcl-B. Together, these data demonstrate that polyubiquitination and proteasomal turnover dictate the expression level and anti-apoptotic capacity of Bcl-B.

Direct molecular weight determination of resin-bound oligopeptides using 252Cf plasma-desorption mass spectrometry.

Plasma-desorption mass spectrometry is proved to be capable of the direct molecular weight determination of totally protected resin-bound oligopeptides. The molecular weight measured is the molecular weight of the fully protected oligopeptide including the molecular weight of the linker, which connects the oligopeptide and the polystyrene resin. This method makes it possible to check the success of (a step in) the synthesis of a peptide before its deprotection or cleaving from the resin. This can make it a strong tool in the analysis of resin-bound oligopeptides.

Identification of the major chlorosomal bacteriochlorophylls of the green sulfur bacteria Chlorobium vibrioforme and Chlorobium phaeovibrioides; their function in lateral energy transfer.

The chlorosomal bacteriochlorophyll (BChl) composition of the green sulfur bacteria Chlorobium vibrioforme and Chlorobium phaeovibrioides was investigated by means of normal-phase high-performance liquid chromatography. From both species a number of homologues was isolated, which were identified by absorption and (252)Cf-plasma desorption mass spectroscopy. Besides BChl d, C. vibrioforme contained a significant amount of BChl c, which may provide an explanation for the previous observation of at least two spectrally different pools of BChl in the chlorosomes of green sulfur bacteria (Otte et al. 1991). C. phaeovibrioides contained various homologues of BChl e only. Absorption spectra in acetone of BChl c, d and e, as well as bacteriopheophytin e are presented. No systematic differences were found for the various homologues of each pigment. In addition to farnesol, the mass spectra revealed the presence of various minor esterifying alcohols in both species, including phytol, oleol, cetol and 4-undecyl-2-furanmethanol, as well as an alcohol of low molecular mass, which is tentatively assumed to be decenol.

Characterization of a synthetic 37-residue fragment of a monoclonal antibody against herpes virus by capillary electrophoresis/electrospray (ionspray) mass spectrometry and 252Cf plasma desorption mass spectrometry.

A peptide comprising 37 amino acids of the antigen binding site of a monoclonal antibody directed against glycoprotein D of herpes simplex virus was synthesized. The synthetic peptide and the impurities formed in the synthesis were characterized by capillary electrophoresis/ionspray mass spectrometry and by 252Cf plasma desorption-time of flight mass spectrometry. The measured average molecular mass of the synthetic peptide was 4627.16 Da, which was only 0.08 Da higher than the calculated value (4627.08 Da). The plasma desorption mass spectrum of the synthetic peptide showed a protonated molecule at m/z 4624.1, which was 4 Da lower than the calculated one (4628.09 Da). The amino acid sequence of the peptide was confirmed in part by electrospray (ionspray) mass spectrometry using a high nozzle skimmer voltage difference. Five impurities were separated and identified by capillary electrophoresis/mass spectrometry and two of them also appeared in the plasma desorption mass spectrum.

Sequence-informative fragmentation of peptides up to a molecular weight of 4.6 kDa in plasma-desorption mass spectrometry.

The potential of plasma-desorption mass spectrometry (PDMS) in peptide sequencing is investigated. This paper shows that PDMS spectra recorded for longer times and using larger amounts of peptide than used for obtaining molecular weight information, provides sequence information on peptides of up to 4.5 kDa molecular weight. This approach strongly enhances the utility of PDMS for validation of the sequence of recombinant peptides.

Human interleukin-3 contains a discontinuous zinc binding domain.

The primary structure of human interleukin-3 contains two amino acid consensus sequences at Glutamate 22- Histidine 26 and Histidine 95-Histidine 98, that are characteristic for zinc binding proteins. Therefore, the hypothesis was tested that human interleukin-3 binds zinc specifically by either one or both sequences. Protein dotblotting, followed by probing with radioactive zinc demonstrated specific zinc binding of interleukin-3. Metal specificity was confirmed by competition experiments with 12 other divalent- and trivalent metal ions. Protease treatment combined with plasma desorption mass spectrometry was used to localize the zinc binding domain. Specific zinc binding was restricted to a fragment composed of Threonine 11-Lysine 28 and Asparagine 80-Lysine 100. It was found to decrease by a factor of five when either of these two amino acid stretches was missing. It is concluded that human interleukin-3 is a zinc binding protein. Interleukin-3 zinc binding capacity is largely determined by both moieties of the protein that contain the consensus sequences. In addition we propose that the zinc binding of hIL-3 is involved in (de)phosphorylation of the hIL-3 receptor.