UBC9 Mutant Reveals the Impact of Protein Dynamics on Substrate Selectivity and SUMO Chain Linkages.

SUMO, a conserved ubiquitin-like protein, is conjugated to a multitude of cellular proteins to maintain genomic integrity and resist genotoxic stress. Studies of the SUMO E2 conjugating enzyme mutant, UBC9P123L, suggested that altered substrate specificity enhances cell sensitivity to DNA damaging agents. Using nuclear magnetic resonance chemical shift studies, we confirm that the mutation does not alter the core globular fold of UBC9, while 15N relaxation measurements demonstrate mutant-induced stabilization of distinct chemical states in residues near the active site cysteine and substrate recognition motifs. We further demonstrate that the P123L substitution induces a switch from the preferential addition of SUMO to lysine residues in unstructured sites to acceptor lysines embedded in secondary structures, thereby also inducing alterations in SUMO chain linkages. Our results provide new insights regarding the impact that structural dynamics of UBC9 have on substrate selection and specifically SUMO chain formation. These findings highlight the potential contribution of nonconsensus SUMO targets and/or alternative SUMO chain linkages on DNA damage response and chemotherapeutic sensitivity.

In the Absence of Central pre-B Cell Receptor Selection, Peripheral Selection Attempts to Optimize the Antibody Repertoire by Enriching for CDR-H3 Y101.

Sequential developmental checkpoints are used to "optimize" the B cell antigen receptor repertoire by minimizing production of autoreactive or useless immunoglobulins and enriching for potentially protective antibodies. The first and apparently most impactful checkpoint requires µHC to form a functional pre-B cell receptor (preBCR) by associating with surrogate light chain, which is composed of VpreB and λ5. Absence of any of the preBCR components causes a block in B cell development that is characterized by severe immature B cell lymphopenia. Previously, we showed that preBCR controls the amino acid content of the third complementary determining region of the H chain (CDR-H3) by using a VpreB amino acid motif (RDR) to select for tyrosine at CDR-H3 position 101 (Y101). In antibodies bound to antigen, Y101 is commonly in direct contact with the antigen, thus preBCR selection impacts the antigen binding characteristics of the repertoire. In this work, we sought to determine the forces that shape the peripheral B cell repertoire when it is denied preBCR selection. Using bromodeoxyuridine incorporation and evaluation of apoptosis, we found that in the absence of preBCR there is increased turnover of B cells due to increased apoptosis. CDR-H3 sequencing revealed that this is accompanied by adjustments to DH identity, DH reading frame, JH, and CDR-H3 amino acid content. These adjustments in the periphery led to wild-type levels of CDR-H3 Y101 content among transitional (T1), mature recirculating, and marginal zone B cells. However, peripheral selection proved incomplete, with failure to restore Y101 levels in follicular B cells and increased production of dsDNA-binding IgM antibodies.

FCRLA-A Resident Endoplasmic Reticulum Protein that Associates with Multiple Immunoglobulin Isotypes in B Lineage Cells.

FCRLA is homologous to receptors for the Fc portion of IgG (FcγR) and is located in the same region of human chromosome one, but has several unusual and unique features. It is a soluble resident ER protein retained in this organelle by unknown mechanisms involving the N-terminal domain, a disordered domain with three Cys residues in close proximity in the human protein. Unlike the FcγRs, FCRLA is not glycosylated and has no transmembrane region. FCRLA is included in this CTMI volume on IgM-binding proteins because it binds IgM in the ER, but quite surprisingly, given the isotype-restricted ligand specificity of the other FcRs, it also binds all other Ig isotypes so far tested, IgG and IgA. In the case of IgM, there is even preferential binding of the secretory and not the transmembrane form. Among B cells, FCRLA is most highly expressed in the germinal center and shows little expression in plasma cells. Based on these observations, we propose that one human FCRLA function is to stop GC B cells from secreting IgM, which would act as a decoy receptor, thus preventing the B cells from capturing antigen, processing it, and presenting the antigen-derived peptides to T follicular helper cells. Without help from these T cells, there would be limited B cell isotype switching, proliferation, and differentiation. On the other hand, FCRLA is downregulated in plasma cells, where IgM secretion is an essential function. FCRLA may also act as a chaperone involved by unknown mechanisms in the proper assembly of Ig molecules of all isotypes.

Soluble membrane attack complex is diagnostic for intraventricular shunt infection in children.

BACKGROUND: Children treated with cerebrospinal fluid (CSF) shunts to manage hydrocephalus frequently develop shunt failure and/or infections, conditions that present with overlapping symptoms. The potential life-threatening nature of shunt infections requires rapid diagnosis; however, traditional microbiology is time consuming, expensive, and potentially unreliable. We set out to identify a biomarker that would identify shunt infection. METHODS: CSF was assayed for the soluble membrane attack complex (sMAC) by ELISA in patients with suspected shunt failure or infection. CSF was obtained at the time of initial surgical intervention. Statistical analysis was performed to assess the diagnostic potential of sMAC in pyogenic-infected versus noninfected patients. RESULTS: Children with pyogenic shunt infection had significantly increased sMAC levels compared with noninfected patients (3,211 ± 1,111 ng/ml vs. 26 ± 3.8 ng/ml, P = 0.0001). In infected patients undergoing serial CSF draws, sMAC levels were prognostic for both positive and negative clinical outcomes. Children with delayed, broth-only growth of commensal organisms (P. acnes, S. epidermidis, etc.) had the lowest sMAC levels (7.96 ± 1.7 ng/ml), suggesting contamination rather than shunt infection. CONCLUSION: Elevated CSF sMAC levels are both sensitive and specific for diagnosing pyogenic shunt infection and may serve as a useful prognostic biomarker during recovery from infection. FUNDING: This work was supported in part by the Impact Fund of Children's of Alabama.

VpreB serves as an invariant surrogate antigen for selecting immunoglobulin antigen-binding sites.

Developmental checkpoints eliminate B cells synthesizing defective immunoglobulin heavy (HC) and light (LC) chains. The first checkpoint tests for formation of a VpreB/λ5/µHC-containing preB-cell receptor (preBCR) and predicts whether µHCs will bind conventional LCs to form membrane IgM. VpreB and λ5 also create a sensing site that interacts with µHC antigen-binding region CDR-H3, but whether it plays a role in immunoglobulin repertoire selection and function is unknown. On a position-by-position basis, we analyzed the amino acid content of CDR-H3s from H chains cloned from living and apoptotic preB cells and from IgG:Antigen structures. Using a panel of DH gene-targeted mice, we show that progressively reducing CDR-H3 tyrosine content increasingly impairs preBCR checkpoint passage. Counting from cysteine at Framework 3 position 96, we found that VpreB particularly selects for tyrosine at CDR-H3 position 101, and that Y101 also binds antigen in IgG:Antigen structures. VpreB thus acts as an early invariant antigen. It selects for particular CDR-H3 amino acids and shapes the specificity of the IgG humoral response. This helps explain why some neutralizing antibodies against pathogens are readily produced while others are rare.