Deciphering the Exact Sequence of Endogenous Soluble B Cell Maturation Antigen and Unbiased Quantitation in Multiple Myeloma Patient Samples by LC-MS.

BACKGROUND: B-cell maturation antigen is a pivotal therapeutic target for multiple myeloma (MM). Membrane-bound BCMA can be cleaved by γ-secretase and shed as soluble BCMA (sBCMA). sBCMA can act as a neutralizing sink to compete with drug, as well as serve as a diagnostic/prognostic biomarker for MM. Antibody-capture based methods, such as enzyme-linked immunosorbent assay (ELISA) and immunoaffinity-liquid chromatography-multiple reaction monitoring (IA-LC-MRM), have been reported and well adopted to measure sBCMA in clinical samples. However, both methods are biased by capturing antibodies. METHODS: We have used various LC-MS workflows to characterize and quantify endogenous sBCMA in MM patient samples, including bottom-up peptide mapping, intact analysis, IA-based, and reagent-free (RF)-LC-MRM quantitation. RESULTS: We have confirmed that sBCMA contains a variable N-terminus and a C-terminus that extends to the transmembrane domain, ending at amino acid 61. Leveraging an in-house synthesized G-1-61 sBCMA recombinant standard, we developed a RF-LC-MRM method for unbiased sBCMA quantitation in MM patient samples. By comparing the results from RF-LC-MRM with ELISA and IA-LC-MRM, we demonstrated that RF-LC-MRM measures a more complete pool of endogenous sBCMA compared to the antibody-based methods. CONCLUSIONS: This work fills the knowledge gap of the exact sequence of endogenous sBCMA for the first time, which differs from the current commercially available standard. Additionally, this work highlights the necessity of identifying the actual sequence of an endogenous soluble target such as sBCMA, both for bioanalytical purposes and to underpin pharmacodynamic measurements.

Mutation in IL36RN impairs the processing and regulatory function of the interleukin-36-receptor antagonist and is associated with DITRA syndrome.

The identification of loss-of-function mutations of the IL36RN gene encoding the interleukin-36 receptor antagonist (IL-36Ra) in generalized pustular psoriasis (GPP) emphasized the key role of this pathway in skin innate immunity and systemic inflammation. It has been previously shown in vitro that removal of the N-terminal amino acid IL36Ra (M1) is critical to its biological activity, but the in vivo contribution of this processing remains unknown. We report herein a new homozygous (c4G>T, pV2F) missense IL36RN mutation segregating in a family with three GPP-affected patients. The V2F mutation does not alter IL-36Ra protein expression but was devoid of any antagonist activity. Mass spectrometry showed that the V2F IL-36Ra mutant retains its first N-terminal methionine. These results provide the first in vivo demonstration that removal of N-terminal methionine of native IL-36Ra is a mandatory step to reach optimal antagonist activity and to prevent sustained skin and systemic inflammation in humans.

Membrane cholesterol modulates STEAP2 conformation during dynamic intracellular trafficking processes leading to broad subcellular distribution.

STEAP2 is a member of the Six-Transmembrane Epithelial Antigen of the Prostate (STEAP) protein family that is proposed to function as metalloreductase. While STEAP2 shows a complex subcellular distribution pattern localizing to both secretory and endocytic pathway organelles, how such broad steady-state distribution is maintained is unknown. Similarly, whether STEAP2 undergoes any compartment-specific modulation during intracellular trafficking has not been reported. Leveraging a newly-identified monoclonal antibody that recognizes a conformation-sensitive epitope nested in the second extracellular loop of STEAP2, we demonstrate that the epitope formation was dependent on the cholesterol content of the membrane in which STEAP2 was embedded. Monitoring the STEAP2-dependent internalization of this antibody uncovered STEAP2's rapid internalization from the cell surface and their subsequence trafficking to the Golgi region and endosome-like puncta. Acute inhibition of endocytosis also increased the detectable amount of STEAP2 at the plasma membrane. Collectively, these experiments demonstrate that an intricate balance of membrane flux between the secretory and endocytic pathways underlies the characteristic broad subcellular localization of STEAP2. By using a cell-based assay that detects the metalloreductase functions of cell surface-localizing STEAP4, STEAP2's metalloreductase activities were not detectable, suggesting that its enzymatic function is suppressed at the plasma membrane. The conformational modulation of STEAP2 by the local membrane cholesterol content can therefore serve as a potential mechanism to modulate STEAP2 function in a compartment-restricted manner, by coupling a pre-existing difference in cholesterol content among different cellular membranes to a dynamic trafficking process leading to broad subcellular distribution.

Molecular basis for the loss-of-function effects of the Alzheimer's disease-associated R47H variant of the immune receptor TREM2.

Triggering receptor expressed on myeloid cells 2 (TREM2) is an immune receptor expressed on the surface of microglia, macrophages, dendritic cells, and osteoclasts. The R47H TREM2 variant is a significant risk factor for late-onset Alzheimer's disease (AD), and the molecular basis of R47H TREM2 loss of function is an emerging area of TREM2 biology. Here, we report three high-resolution structures of the extracellular ligand-binding domains (ECDs) of R47H TREM2, apo-WT, and phosphatidylserine (PS)-bound WT TREM2 at 1.8, 2.2, and 2.2 Å, respectively. The structures reveal that Arg47 plays a critical role in maintaining the structural features of the complementarity-determining region 2 (CDR2) loop and the putative positive ligand-interacting surface (PLIS), stabilizing conformations capable of ligand interaction. This is exemplified in the PS-bound structure, in which the CDR2 loop and PLIS drive critical interactions with PS via surfaces that are disrupted in the variant. Together with in vitro and in vivo characterization, our structural findings elucidate the molecular mechanism underlying loss of ligand binding, putative oligomerization, and functional activity of R47H TREM2. They also help unravel how decreased in vitro and in vivo stability of TREM2 contribute to loss of function in disease.

Auto-induction of Pichia pastoris AOX1 promoter for membrane protein expression.

Pichia pastoris is a highly successful recombinant protein expression system due to its ability to quickly generate large quantities of recombinant proteins in simple media. P. pastoris has been used to successfully generate milligram quantities of many important human membrane proteins, including G-protein coupled receptors, ion channels, and transporters, which are becoming increasingly important therapeutic targets. Despite these successes, protein expression in P. pastoris is still cumbersome due to a need to change growth media from glycerol media to methanol induction media, which minimizes inhibition of the AOX1 promoter by residual glycerol. Taking advantage of this behavior of the AOX1 promoter, we developed Buffered extra-YNB Glycerol Methanol (BYGM) auto-induction media (100 mM potassium phosphate pH 6.0, 2.68% w/v YNB, 0.4% v/v glycerol, 0.5% v/v methanol, and 8 × 10-5% w/v biotin) which not only simplified the protein expression process, but also optimized protein expression levels in P. pastoris. We successfully used this auto-induction method to overexpress the target in both MutS and Mut+ strains. Moreover, we show that this method can facilitate screening high-expressing clones, as well as enable parallel protein production in P. pastoris.

A rare IL33 loss-of-function mutation reduces blood eosinophil counts and protects from asthma.

IL-33 is a tissue-derived cytokine that induces and amplifies eosinophilic inflammation and has emerged as a promising new drug target for asthma and allergic disease. Common variants at IL33 and IL1RL1, encoding the IL-33 receptor ST2, associate with eosinophil counts and asthma. Through whole-genome sequencing and imputation into the Icelandic population, we found a rare variant in IL33 (NM_001199640:exon7:c.487-1G>C (rs146597587-C), allele frequency = 0.65%) that disrupts a canonical splice acceptor site before the last coding exon. It is also found at low frequency in European populations. rs146597587-C associates with lower eosinophil counts (ß = -0.21 SD, P = 2.5×10-16, N = 103,104), and reduced risk of asthma in Europeans (OR = 0.47; 95%CI: 0.32, 0.70, P = 1.8×10-4, N cases = 6,465, N controls = 302,977). Heterozygotes have about 40% lower total IL33 mRNA expression than non-carriers and allele-specific analysis based on RNA sequencing and phased genotypes shows that only 20% of the total expression is from the mutated chromosome. In half of those transcripts the mutation causes retention of the last intron, predicted to result in a premature stop codon that leads to truncation of 66 amino acids. The truncated IL-33 has normal intracellular localization but neither binds IL-33R/ST2 nor activates ST2-expressing cells. Together these data demonstrate that rs146597587-C is a loss of function mutation and support the hypothesis that IL-33 haploinsufficiency protects against asthma.

Topogenesis and cell surface trafficking of GPR34 are facilitated by positive-inside rule that effects through a tri-basic motif in the first intracellular loop.

Protein folding, topogenesis and intracellular targeting of G protein-coupled receptors (GPCRs) must be precisely coordinated to ensure correct receptor localization. To elucidate how different steps of GPCR biosynthesis work together, we investigated the process of membrane topology determination and how it relates to the acquisition of cell surface trafficking competence in human GPR34. By monitoring a fused FLAG-tag and a conformation-sensitive native epitope during the expression of GPR34 mutant panel, a tri-basic motif in the first intracellular loop was identified as the key topogenic signal that dictates the orientation of transmembrane domain-1 (TM1). Charge disruption of the motif perturbed topogenic processes and resulted in the conformational epitope loss, post-translational processing alteration, and trafficking arrest in the Golgi. The placement of a cleavable N-terminal signal sequence as a surrogate topogenic determinant overcame the effects of tri-basic motif mutations and rectified the TM1 orientation; thereby restored the conformational epitope, post-translational modifications, and cell surface trafficking altogether. Progressive N-tail truncation and site-directed mutagenesis revealed that a proline-rich segment of the N-tail and all four cysteines individually located in the four separate extracellular regions must simultaneously reside in the ER lumen to muster the conformational epitope. Oxidation of all four cysteines was necessary for the epitope formation, but the cysteine residues themselves were not required for the trafficking event. The underlying biochemical properties of the conformational epitope was therefore the key to understand mechanistic processes propelled by positive-inside rule that simultaneously regulate the topogenesis and intracellular trafficking of GPR34.

Overexpression of cryoglobulin-like single-chain antibody induces morular cell phenotype via liquid-liquid phase separation in the secretory pathway organelles.

Cryoprecipitation of immunoglobulins is often reported in association with B-cell lymphoproliferative disorders and plasma cell dyscrasias. However, the biochemical basis of such cryoglobulin behaviors is not well understood because of a general lack of suitable experimental systems. Here, we report the identification and characterization of a single-chain antibody (scFv-Fc) that recapitulates cryoglobulin-like properties. When model scFv-Fc protein was engineered to multimerize, by appending the secretory tailpiece (stp) of human immunoglobulin µ-chain to the C terminus, the resulting oligomeric scFv-Fc-stp protein acquired two unexpected properties: the induction of a morular cell phenotype during protein biosynthesis and the cryoprecipitation of secreted proteins in harvested cell culture media. The turbidity of the culture media and the inclusion bodies that gave morular appearances were attributed to microscopic spherical protein droplet formation, a hallmark characteristic of liquid-liquid phase separation (LLPS) event. Mutagenesis approaches revealed that these two phenomena were independent of covalent protein oligomerization induced by stp. Disruption of the N-linked glycosylation motif in the stp region enhanced morular phenotype propensity but reduced protein secretion. Intermolecular disulfide bonds that stabilize Fc dimers and oligomers were necessary for efficient induction of LLPS, but their simultaneous elimination could not abrogate the LLPS propensity completely. Noncovalent protein-protein interactions between scFv-Fc-stp chains sufficiently established a basis for LLPS induction. Morular cell phenotypes and cryoprecipitation were clearly underpinned by intrinsic physicochemical properties embedded in the overexpressed cargo protein. Overproduction of condensation-prone secretory proteins that culminate in LLPS in the endoplasmic reticulum therefore serves as a path to produce morular Russell body phenotype.

Russell body phenotype is preferentially induced by IgG mAb clones with high intrinsic condensation propensity: relations between the biosynthetic events in the ER and solution behaviors in vitro.

The underlying reasons for why some mAb (monoclonal antibody) clones are much more inclined to induce a Russell body (RB) phenotype during immunoglobulin biosynthesis remain elusive. Although RBs are morphologically understood as enlarged globular aggregates of immunoglobulins deposited in the endoplasmic reticulum (ER), little is known about the properties of the RB-inducing mAb clones as secretory cargo and their physical behaviors in the extracellular space. To elucidate how RB-inducing propensities, secretion outputs, and the intrinsic physicochemical properties of individual mAb clones are interrelated, we used HEK293 cells to study the biosynthesis of 5 human IgG mAbs for which prominent solution behavior problems were known a priori. All 5 model mAbs with inherently high condensation propensities induced RB phenotypes both at steady state and under ER-to-Golgi transport block, and resulted in low secretion titer. By contrast, one reference mAb that readily crystallized at neutral pH in vitro produced rod-shaped crystalline bodies in the ER without inducing RBs. Another reference mAb without notable solution behavior issues did not induce RBs and was secreted abundantly. Intrinsic physicochemical properties of individual IgG clones thus directly affected the biosynthetic steps in the ER, and thereby produced distinctive cellular phenotypes and influenced IgG secretion output. The findings implicated that RB formation represents a phase separation event or a loss of colloidal stability in the secretory pathway organelles. The process of RB induction allows the cell to preemptively reduce the extracellular concentration of potentially pathogenic, highly aggregation-prone IgG clones by selectively storing them in the ER.

Modulation of in vivo IgG crystallization in the secretory pathway by heavy chain isotype class switching and N-linked glycosylation.

Crystalline bodies (CBs) can develop in the endoplasmic reticulum (ER) of antibody-producing cells. Although this phenotype is often reported in association with plasma cell dyscrasias and other hematological disorders, the details of CB biogenesis and CB's roles in pathophysiology remain poorly understood. Using an imaging-based screening method, we identified a secretion-competent human IgG2/λ clone that develops spindle-shaped intracellular crystals in transiently-transfected HEK293 cells upon Brefeldin A treatment. When stably overexpressed from CHO cells, the IgG2/λ clone spontaneously produced spindle-shaped CBs in the ER. Some CBs were released to the extracellular space while remaining enclosed by the membranes of secretory pathway origin. Structural modeling on the variable-region did not uncover prominent surface characteristics such as charge clusters. In contrast, alterations to the constant domain-encoded properties revealed their modulatory roles in CB-inducing propensities and CB morphology. For example, deletion of the entire Fc domain changed the morphology of CBs into thin filaments. Elimination of an N-linked glycan by a N297A mutation promoted Russell body biogenesis accompanied by marked reduction in IgG secretion. Isotype class switching from the original IgG2 to IgG1 and IgG4 changed the crystal morphology from spindle-shaped to long needle and acicular shaped, respectively. The IgG3 version, in contrast, suppressed the CB formation. Either the HC or LC alone or the Fc-domain alone did not trigger CB biogenesis. An IgG's in vivo crystal morphology and crystallization propensity can thus be modulated by the properties genetically and biochemically encoded in the HC constant region.

Anti-tumor effect of CT-322 as an adnectin inhibitor of vascular endothelial growth factor receptor-2.

CT-322 is a new anti-angiogenic therapeutic agent based on an engineered variant of the tenth type III domain of human fibronectin, i.e., an Adnectin™, designed to inhibit vascular endothelial growth factor receptor (VEGFR)-2. This PE Gylated Adnectin was developed using an mRNA display technology. CT-322 bound human VEGFR-2 with high affinity (K(D), 11 nM), but did not bind VEGFR-1 or VEGFR-3 at concentrations up to 100 nM, as determined by surface plasmon resonance studies. Western blot analysis showed that CT-322 blocked VEGF-induced phosphorylation of VEGFR-2 and mitogen-activated protein kinase in human umbilical vascular endothelial cells. CT-322 significantly inhibited the growth of human tumor xenograft models of colon carcinoma and glioblastoma at doses of 15-60 mg/kg administered 3 times/week. Anti-tumor effects of CT-322 were comparable to those of sorafenib or sunitinib, which inhibit multiple kinases, in a colon carcinoma xenograft model, although CT-322 caused less overt adverse effects than the kinase inhibitors. CT-322 also enhanced the anti-tumor activity of the chemotherapeutic agent temsirolimus in the colon carcinoma model. The high affinity and specificity of CT-322 binding to VEGFR-2 and its anti-tumor activities establish CT-322 as a promising anti-angiogenic therapeutic agent. Our results further suggest that Adnectins are an important new class of targeted biologics that can be developed as potential treatments for a wide variety of diseases.

A diversity of antibody epitopes can induce signaling through the erythropoietin receptor.

Stimulation of red cell production through agonism of the erythropoietin receptor (EpoR) has historically been accomplished through administration of erythropoietin (EPO), the native ligand. The short half-life of EPO has led to the development of a variety of other agonists, including antibodies. It is of considerable interest to understand how these agents might activate the EpoR and whether or not it is important to bind in a manner similar to the native ligand. The binding epitopes of a panel of eight agonistic, single-chain antibody (scFv-Fc) constructs were determined through scanning alanine mutagenesis as well as more limited arginine mutagenesis of the receptor. It was found that while some of these constructs bound to receptor epitopes shared by the ligand, others bound in completely unique ways. The use of a panel of agonists and scanning mutagenesis can define the critical binding regions for signaling; in the case of the EpoR, these regions were remarkably broad.

Directed evolution of high-affinity antibody mimics using mRNA display.

We constructed a library of >10(12) unique, covalently coupled mRNA-protein molecules by randomizing three exposed loops of an immunoglobulin-like protein, the tenth fibronectin type III domain (10Fn3). The antibody mimics that bound TNF-alpha were isolated from the library using mRNA display. Ten rounds of selection produced 10Fn3 variants that bound TNF-alpha with dissociation constants (K(d)) between 1 and 24 nM. After affinity maturation, the lowest K(d) measured was 20 pM. Selected antibody mimics were shown to capture TNF-alpha when immobilized in a protein microarray. 10Fn3-based scaffold libraries and mRNA-display allow the isolation of high-affinity, specific antigen binding proteins; potential applications of such binding proteins include diagnostic protein microarrays and protein therapeutics.