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Research Article

A peptide extension dictates IgM assembly

Dzana Pasalic, Benedikt Weber, Chiara Giannone, Tiziana Anelli, Roger Müller, Claudio Fagioli, Manuel Felkl, Christine John, Maria Francesca Mossuto, View ORCID ProfileChristian F. W. Becker, Roberto Sitia, and Johannes Buchner
PNAS first published September 27, 2017; https://doi.org/10.1073/pnas.1701797114
Dzana Pasalic
aCenter for Integrated Protein Science Munich at the Department Chemie, Technische Universität München, 85748 Garching, Germany;
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Benedikt Weber
aCenter for Integrated Protein Science Munich at the Department Chemie, Technische Universität München, 85748 Garching, Germany;
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Chiara Giannone
bDivision of Genetics and Cell Biology, Università Vita-Salute IRCCS Ospedale San Raffaele, 20132 Milano, Italy;
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Tiziana Anelli
bDivision of Genetics and Cell Biology, Università Vita-Salute IRCCS Ospedale San Raffaele, 20132 Milano, Italy;
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Roger Müller
aCenter for Integrated Protein Science Munich at the Department Chemie, Technische Universität München, 85748 Garching, Germany;
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Claudio Fagioli
bDivision of Genetics and Cell Biology, Università Vita-Salute IRCCS Ospedale San Raffaele, 20132 Milano, Italy;
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Manuel Felkl
cFakultät Chemie, Institut für Biologische Chemie, Universität Wien, 1090 Wien, Austria
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Christine John
aCenter for Integrated Protein Science Munich at the Department Chemie, Technische Universität München, 85748 Garching, Germany;
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Maria Francesca Mossuto
bDivision of Genetics and Cell Biology, Università Vita-Salute IRCCS Ospedale San Raffaele, 20132 Milano, Italy;
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Christian F. W. Becker
cFakultät Chemie, Institut für Biologische Chemie, Universität Wien, 1090 Wien, Austria
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  • ORCID record for Christian F. W. Becker
Roberto Sitia
bDivision of Genetics and Cell Biology, Università Vita-Salute IRCCS Ospedale San Raffaele, 20132 Milano, Italy;
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  • For correspondence: sitia.roberto@hsr.it johannes.buchner@tum.de
Johannes Buchner
aCenter for Integrated Protein Science Munich at the Department Chemie, Technische Universität München, 85748 Garching, Germany;
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  • For correspondence: sitia.roberto@hsr.it johannes.buchner@tum.de
  1. Edited by Linda L. Randall, University of Missouri-Columbia, Columbia, MO, and approved September 1, 2017 (received for review February 1, 2017)

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  • Fig. 1.
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    Fig. 1.

    Effects of μtp alanine replacements on IgM polymerization in cultured cells. (A) Cell SNs were collected after 4 h of incubation in MEM and resolved by nonreducing gels, and blots were decorated with Ig-μ antibodies. Owing to unbalanced μ-L stoichiometries, the relative amounts of μ, μ-L, μ2-L, and μ2-L2 assemblies varied in different transfectants. (B) Different gel mobility of mutants secreted as μ2-L2 is evident under reducing (a) and nonreducing (b) conditions.

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    Fig. S1.

    Characterization of IgM alanine mutants. (A) Sucrose gradient fractionation assays confirm that mutants with reduced hydrophobicity are secreted as μ2-L2 subunits. Cell SNs were processed as described in Materials and Methods. Eight of the total 12 fractions (FR.) collected were resolved by nonreducing polyacrylamide gradient gels. Polymeric wt IgM accumulates in fractions 7–8, while Y562, V564, L566, and I567 mutants are present mostly in fractions 2–4. These results exclude the formation of polymers. Note that the M568A mutant yields both polymers and monomers. (B) Cell lysates were collected after 4 h of incubation in MEM and resolved by nonreducing gels, and blots were decorated with Ig-μ (Top) or Ig-λ (Bottom) antibodies. Different assembly intermediates (μ, μ-L, μ2-L, and μ2-L2) are indicated on the right-hand margin. The top two bands are hexamers and pentamers. IgM Std, IgM standard.

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    Fig. S2.

    Mutants with decreased hydrophobicity do not bind J chains. HEK-293T cells stably expressing l chains were cotransfected with vectors encoding murine J chain and the indicated μ-chain mutants. Aliquots of the SNs were resolved under nonreducing conditions, and the blot was sequentially decorated with anti-J and anti-μ antibodies (6).

  • Fig. 2.
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    Fig. 2.

    Kinetics of Cμ4tp oligomerization in vitro. Cμ4tp was refolded and purified as described in Materials and Methods. SEC fractions containing the Cμ4tp monomer were pooled, and the concentration was adjusted to 1 mg/mL. Aliquots were stored at 4 °C and analyzed at the indicated time points. (A, a) Representative SE-HPLC profiles on a Superdex 200 10/300 GL in PBS at 20 °C. For each time point, 100 μg of sample was injected. A BioRad gel filtration standard (gray) was used as a size reference, where peak 2, which is the closest to the monomer, refers to myoglobin (horse) with a molecular mass of 17 kDa. (A, b) Monomer (red), dimer (orange), and dodecamer (green) formation is shown over time. Error bars indicate the SD of three independent SE-HPLC measurements. (B) SDS/PAGE analysis under nonreducing conditions of the kinetic samples blocked with AMS at indicated time points. Relative optical (Opt.) density of the monomer (red bar) and covalent dimer (orange bar) bands over time is shown. Error bars represent the SD of the optical density of three individual nonreducing SDS/PAGE experiments.

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    Fig. S3.

    Effect of the redox system during oligomerization. Cμ4tp was refolded under the standard GSSG/GSH ratio. The monomer was isolated as outlined above, and the protein concentration was adjusted to 1 mg/mL. The sample was then dialyzed “ON” against PBS containing three different ratios of the redox system. Aliquots were stored at 4 °C and analyzed at the indicated time points by SE-HPLC on a Superdex 200 10/300 GL in PBS at 20 °C. For each time point, 100 μg of sample was injected. Black bars indicate no GSSG/GSH, yellow bars indicate GSSG/GSH = 1 mM/0.5 Mm, gray bars indicate GSSG/GSH = 1 mM/1 Mm, and green bars indicate GSSG/GSH = 0.5 mM/1 mM.

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    Fig. 3.

    Essential role of C575 disulfides in Cμ4tp oligomerization in vitro. (A and B) Effects of AMS on Cμ4tp oligomerization. (A) AMS (10 mM final) was added to freshly isolated monomeric Cμ4tp, and the sample was concentrated to 15 mg/mL. (B) AMS was added after 0 (black), 1 (red), 2 (blue), 3 (pink), 6 (green), or 9 (purple) d of incubation at room temperature, and the samples were analyzed at the indicated time points. (C) Defective oligomerization of C575 Cμ4tp mutants: C575S and C575A monomer was isolated and concentrated to 1 mg/mL (red) or 15 mg/mL (black) directly in PBS or after dialysis against PBS with 1 M NaCl. (D) Representative SE-HPLC experiments show the disruption of the C575 disulfide bonds by DTT, which causes (Cμ4tp2)6 dissociation. Dodecamers were obtained by processing the concentrated Cμ4tp over the preparative SE column. Fractions containing (Cμ4tp2)6 were pooled, the protein concentration was adjusted to 1 mg/mL, and DTT (1 mM final) was added. Samples were analyzed at the indicated time points. Green bars indicate (Cμ4tp2)6, orange bars indicate Cμ4tp2, and red bars indicate Cμ4tp. All SE-HPLC runs were performed injecting 100 μg of sample on a Superdex 200 10/300 GL column at 20 °C. Error bars represent the SD of three independent SE-HPLC measurements.

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    Fig. 4.

    Oligomerization ability of Cμ4tp alanine mutants in vitro. (A) Schematic representation of their oligomerization ability in vitro. Mutants were refolded, and monomers were isolated as previously described. To accelerate oligomerization, monomers were concentrated to 15 mg/mL and then run over a preparative SE column. Fractions containing the oligomer were pooled, and the concentration was adjusted to 0.5 mg/mL for analytical ultracentrifugation (aUC) and to 1 mg/mL for SEC-MALS analysis. When no oligomers formed, fractions containing monomer and dimers were pooled and analyzed at the same concentrations. For mutants able to oligomerize the height of the dodecamer, the peak was normalized with respect to that of the wt dodecamer peak. Red indicates Cμ4tp, orange indicates Cμ4tp2, yellow indicates (Cμ4tp2)5, and green indicates (Cμ4tp2)6. (B) SEC-MALS (a) and aUC (b) profiles of nonoligomerizing mutants in PBS. c(s), concentration of species as a function of sedimentation; Sed. coefficient, sedimentation coefficient. (C) Schematic representation of the region prone to β-aggregation predicted by TANGO and by AGGRESCAN of the wt μtp peptide and 18 alanine μtp mutants.

  • Fig. S4.
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    Fig. S4.

    Oligomerization ability of Cμ4tp alanine mutants in vitro. (A, a and b) SEC-MALS (Left) and the aUC profiles of oligomerizing mutants in PBS (Right). (A, c) Chromatograms of the preparative SEC of aggregating mutants. aUC, analytical ultracentrifugation; Sed. coeffcient, sedimentation coefficient. (B) Oligomerization ability of nonoligomerizing mutants at 15 mg/mL. The samples were analyzed on SE-HPLC injecting 100 μg of protein. (C) TANGO and AGGRESCAN scores for aggregation propensity of μtp mutants.

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    Fig. 5.

    Characterization of wt and mutant μtp peptides. (A) Oligomerization ability of the wt μtp peptide. SEC profiles of μtp peptides resuspended before (a) or after (b) oxidation on a Superdex 75 10/300 GL column on day 0 (red) and day 3 (black) after resuspension/oxidation are shown. In a, the peptide was resuspended in Tris/EDTA buffer (pH 7.5) to a final concentration of 0.4 mg/mL (207 μM) and stored at room temperature (RT). In b, a redox system was added (1 mM GSSG, 0.5 mM GSH final) and the sample was incubated for 2 h at RT under stirring. Glutathione was then removed by centrifugation/filtration and subsequent dilution with Tris/EDTA buffer. Samples were stored at RT. (B and C) Secondary structure characterization of resuspended/oxidized wt μtp within the far-UV range. (B, a) Overlay of the CD spectra of the resuspended (green) and oxidized (red) wt peptide. Spectra were acquired at 20 °C. (B, b) PSIPRED prediction of the wt μtp secondary structure. (C) Overlay of the CD spectra of four μtp mutants after oxidation. Red indicates wt, black indicates Y562A, blue indicates V564A, pink indicates L566A, and green indicates I567A.

  • Fig. 6.
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    Fig. 6.

    Role of the C-terminal part of the μtp in cell culture and in vitro polymerization. (A, a and b) Secreted IgM was collected from the indicated mutants and analyzed as described in the legend for Fig. 1. (B) In vitro oligomerization of Cμ4tp mutants. (Left) SEC-MALS profiles of isolated oligomers at 1 mg/mL in PBS. (Right) Analytical ultracentrifugation (aUC) profiles at 0.5 mg/mL in PBS. (a) Red represents wt, and black represents ΔY576. (b) Red represents wt, black represents Y576G, cyan represent Y576A, purple represents Y576W, and orange represents Y576F. (c) Red represents wt, blue represents Y18K, and yellow represents Y18D. (d) Red represents wt, and black represents C575YY576C. Sed. coefficient, sedimentation coefficient.

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    Fig. 7.

    Models for IgM polymerization. (A) Model for Cμ4tp oligomerization in vitro. (a and b) μtp (dark blue tail) conformational changes in Cμ4tp induced by the formation of a C575 disulfide bond (yellow circles). In a, two different possible tailpiece conformations are depicted. Cμ4tp dimerization is coupled to oligomerization (b and c), leading to a hexamer (d). Noncovalent interactions might occur either between two adjacent μtps or between the μtp and the Cμ4 domain in trans. (B) Assembly of IgM in living cells. Cysteines involved in interdomain disulfide bridges are indicated by yellow circles. (a) Fc region of two μ-L “hemimers” is shown with one of the two μtp arrangements shown in A. Covalent and noncovalent interactions link C337 and surrounding regions in the Cμ2 domains (depicted here in green), limiting the mobility of the C-terminal portions, and hence favoring collisions between pairs of Cμ4 and μtps, to produce a μ2-L2 as depicted in b. Antigen-binding (Fab) fragments attached to the Cμ2 domains were deleted for clarity. (b) Snippet of the IgM Fc dodecamer. Formation of the C575 disulfide results in conformational modifications the Cμ4tp domain, allowing the establishment of noncovalent interactions (red boxes) either between two adjacent μtps or between the Cμ4 and a μtp in trans. The Cμ3 domains in adjacent subunits must become close enough for C414 to form a disulfide bond, a feature favored in hexamers. It is noteworthy that C414 does not form intersubunit disulfides in membrane IgM.

Tables

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    Table 1.

    Nonoligomerizing, oligomerizing, and aggregating Cμ4tp mutants with the molecular mass and S value of the biggest species formed

    MutantOligomer sizeMM, kDaS value
    WT12×2018.5
    C575A1×141.7
    Y562A2×282.5
    V564A2×292.5
    L566A2×282.5
    I567A2×282.6
    P559A12×2118.5
    T560A12×1928.4
    L561A12×1958.4
    S564A12×1797.7
    M568A10×1687.5
    S569A12×1918.1
    T571A12×1988.4
    G572A12×1978.2
    G573A12×1938.3
    T574A12×2008.5
    Y576A12×1918.6
    N563AAggregates——
    D570AAggregates——
    • MM and S value were determined by SEC-MALS and aUC, respectively. aUC, analytical ultracentrifugation; MM, molecular mass.

    • View popup
    Table 2.

    Synthetic μtp peptides

    SequenceNameMMcalc, DaMMobs, DaPurity, %
    PTLYNVSLIMSDTGGTCYwt μtp1,935.21,934.196
    PTLYNVSLIMSDTGGTSYC575S1,919.11,918.593
    PTLYNVSLIMSDTGGTAYC575A1,903.11,901.794
    PTLANVSLIMSDTGGTCYY562A1,843.11,842.090
    PTLYNASLIMSDTGGTCYV564A1,907.11,906.789
    PTLYNVSAIMSDTGGTCYL566A1,893.11,892.191
    PTLYNVSLAMSDTGGTCYI567A1,893.11,892.292
    • All mutations are highlighted in bold, amino acids double-coupled during synthesis are indicated in italics, and pseudoproline building blocks are underlined. MMcalc, calculated molecular mass; MMobs, observed molecular mass.

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Peptide extension dictates IgM assembly
Dzana Pasalic, Benedikt Weber, Chiara Giannone, Tiziana Anelli, Roger Müller, Claudio Fagioli, Manuel Felkl, Christine John, Maria Francesca Mossuto, Christian F. W. Becker, Roberto Sitia, Johannes Buchner
Proceedings of the National Academy of Sciences Sep 2017, 201701797; DOI: 10.1073/pnas.1701797114

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Peptide extension dictates IgM assembly
Dzana Pasalic, Benedikt Weber, Chiara Giannone, Tiziana Anelli, Roger Müller, Claudio Fagioli, Manuel Felkl, Christine John, Maria Francesca Mossuto, Christian F. W. Becker, Roberto Sitia, Johannes Buchner
Proceedings of the National Academy of Sciences Sep 2017, 201701797; DOI: 10.1073/pnas.1701797114
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