Journal: Nature Structural & Molecular Biology

Article Title: Structure of the decoy module of human glycoprotein 2 and uromodulin and its interaction with bacterial adhesin FimH

doi: 10.1038/s41594-022-00729-3

Figure Lengend Snippet: a , Domain architecture of mature human GP2 and UMOD. Domains are indicated by their acronyms, except for UMOD epidermal growth factor (EGF) domains that are labeled according to their roman number, the single EGF domain of GP2 (corresponding to UMOD EGF IV) that is labeled as ‘E’ and the β-hairpin of the decoy module (‘β’). The UMOD D10C epitope recognized by Bence-Jones proteins (BJP) is shown as a green stripe. Black and magenta inverted tripods indicate the N -glycosylation sites of GP2 and UMOD, respectively, with the high-mannose chains attached to GP2 N65 (this study) and UMOD N275 , colored cyan. The position corresponding to the alternative 3’ splice site generating the β isoform of GP2 (T178 | D179) and the elastase cleavage site of UMOD (S291 | S292) are indicated by vertical blue and orange arrows, respectively. b , Alignment of D10C domain sequences from human (h) and murine (m) homologues of GP2 and UMOD, as well as liver-specific zona pellucida protein (LZP/OIT3, a molecule that can also interact with UMOD in the kidney and urine ) and von Willebrand factor D and EGF domain-containing protein (VWDE; a protein involved in appendage regeneration in a variety of vertebrate species ). Identical residues are highlighted in white and shaded in red; conserved residues are red and marked by blue frames when clustered. Consensuses at different sequence identity thresholds, based on a comprehensive alignment of homologous sequences, are also reported (bold uppercase characters: amino acids with the same one-letter code; regular lowercase characters: l, [I,V,L]; h, [F,Y,W,H,I,V,L]; + , [H,K,R]; -, [D,E]; p, [Q,N,S,T,C,H,K,R,D,E]; u, [G,A,S]; s, [G,A,S,V,T,D,N,P,C]; t, [G,A,S,Q,N,S,T,C,H,K,R, D,E]; (.), any amino acid). GP2 secondary structure elements, rainbow-colored from blue (N-terminus) to red (C-terminus), and disulfide bond connectivity are shown above and below the alignment, respectively. Other elements are labeled as in (a), with a green box indicating the BJP epitope . Black bold numbers above the alignment indicate hGP2 residues; light grey numbers between parentheses refer to the corresponding hUMOD residues. c , Cartoon representation of the GP2 decoy module, rainbow-colored following the same scheme used for the secondary structure elements of (b). Disulfide bonds are represented as grey sticks. d , Topology and disulfide connectivity diagram of the decoy module.

Article Snippet: A corresponding gene and an equivalent UMOD construct, as well as GP2 Δ31-59, Δ31-88 and N65A mutant genes, were also synthesized (GenScript) and all constructs were cloned into pLJ6, a mammalian expression vector derived from pHLsec3 (ref. ).

Techniques: Labeling, Glycoproteomics, Sequencing

Journal: Nature Structural & Molecular Biology

Article Title: Structure of the decoy module of human glycoprotein 2 and uromodulin and its interaction with bacterial adhesin FimH

doi: 10.1038/s41594-022-00729-3

Figure Lengend Snippet: a , For assessing whether the lectin domain of FimH is able to bind in vitro to the branch of GP2 or the equivalent region of UMOD (corresponding to the respective decoy modules, see main text), untagged FimH L was expressed in E. coli and a crude periplasmic extract was prepared. n = 2. b , SEC analysis of the material eluted after incubating purified His-tagged GP2 or UMOD decoy modules bound to IMAC beads with the FimH L -containing E. coli periplasmic extract (magenta curves). In both cases, reducing SDS-PAGE of peak fractions and tandem mass spectrometry (MS/MS) of the corresponding ~15 kDa bands show the presence of complexes between the decoy modules and the bacterial adhesin, indicating that the former are able to selectively recognize the latter among the pool of periplasmic proteins. SEC elution profiles of the GP2 and UMOD decoy domains by themselves are also shown (light blue curves), and a low-molecular weight contaminant peak is indicated by *. GP2 decoy module, UMOD decoy module: n = 3; GP2 decoy module/FimH L , UMOD decoy module/FimH L , n = 2. c , Control SEC profile of unbound His-tagged FimH L with SDS-PAGE analysis of the peak. § indicates minor high-molecular weight contaminants eluting with or close to the void volume. n = 3.

Article Snippet: A corresponding gene and an equivalent UMOD construct, as well as GP2 Δ31-59, Δ31-88 and N65A mutant genes, were also synthesized (GenScript) and all constructs were cloned into pLJ6, a mammalian expression vector derived from pHLsec3 (ref. ).

Techniques: In Vitro, Purification, SDS Page, Mass Spectrometry, Tandem Mass Spectroscopy, Molecular Weight, Control, High Molecular Weight

Journal: Nature Structural & Molecular Biology

Article Title: Structure of the decoy module of human glycoprotein 2 and uromodulin and its interaction with bacterial adhesin FimH

doi: 10.1038/s41594-022-00729-3

Figure Lengend Snippet: a , Overall structure of the GP2 branch region/decoy module, depicted in cartoon representation with β-strands in blue, 3 10 helices in cyan and loops in light gray. Disulfides and glycans are shown as yellow and dark gray sticks, respectively, with oxygen atoms in red and nitrogen atoms in blue. b , Reducing western blot comparison of the expression and secretion of GP2 constructs corresponding to the entire branch, D10C or D8C. n = 3. c – g , Details of the GP2 structure rationalize the effect of kidney disease-associated UMOD mutations affecting a set of residues identical between the two proteins (Supplementary Table ). Selected GP2 D10C domain residues and mutations affecting the corresponding identical residues of UMOD are as follows: GP2 D61, P62, C 1 63→UMOD D172H, P173L/R, C174R ( c ); GP2 R74, C 85, D86, C 4 113, C 10 177→UMOD R185C/G/H/L/S, C195F/Y, D196N/Y, C223R/Y, C287F ( d ); GP2 P62, C 1 63, W92, C 8 157, V163→UMOD P173L/R, C174R, W202C/S, C267F, V273F/L ( e ); GP2 C 1 63, R94, C 8 157→UMOD C174R, R204G/P, C267F ( f ); GP2 Y164, C 10 177→UMOD Y274C/H, C287F ( g ).

Article Snippet: A corresponding gene and an equivalent UMOD construct, as well as GP2 Δ31-59, Δ31-88 and N65A mutant genes, were also synthesized (GenScript) and all constructs were cloned into pLJ6, a mammalian expression vector derived from pHLsec3 (ref. ).

Techniques: Western Blot, Comparison, Expressing, Construct

Journal: Nature Structural & Molecular Biology

Article Title: Structure of the decoy module of human glycoprotein 2 and uromodulin and its interaction with bacterial adhesin FimH

doi: 10.1038/s41594-022-00729-3

Figure Lengend Snippet: a-b , GP2 D10C residues corresponding to UMOD amino acids mutated in kidney disease patients (panel a, red) are largely clustered into two highly conserved protein regions (panel b). Sequence conservation is represented using a color spectrum ranging from green (lowest conservation) to violet (highest conservation). c-g , Alternative representation of the structural details shown in Fig. , with residues colored by sequence conservation.

Article Snippet: A corresponding gene and an equivalent UMOD construct, as well as GP2 Δ31-59, Δ31-88 and N65A mutant genes, were also synthesized (GenScript) and all constructs were cloned into pLJ6, a mammalian expression vector derived from pHLsec3 (ref. ).

Techniques: Sequencing

Journal: Nature Structural & Molecular Biology

Article Title: Structure of the decoy module of human glycoprotein 2 and uromodulin and its interaction with bacterial adhesin FimH

doi: 10.1038/s41594-022-00729-3

Figure Lengend Snippet: Supporting MS2 spectrum of precursor m/z 1170.46, 61 DPCQNYTLL 69 , carrying oligomannose-5 (HexNAc2Hex5). Prepared by Asp-N digestion of the GP2 branch purified from HEK293T cells. N-glycan structures are depicted following the Consortium for Functional Glycomics (CFG) notation: HexNAc, N-acetylglucosamine (blue square); Hex, mannose (green circle). The cysteine residue is carbamidomethylated. Detected peptide-backbone fragment ions are presented in the peptide sequence. Interestingly, complex-type carbohydrate structures were also found to be attached to N65. This is consistent with the observation that, although UMOD N275 and GP2 N65 are both located in the groove between the β-hairpin and the D10C domain of the respective decoy modules, N65 is relatively more exposed than N275 in the structure (Extended Data Fig. ), making the N65 glycan chains more susceptible to modification.

Article Snippet: A corresponding gene and an equivalent UMOD construct, as well as GP2 Δ31-59, Δ31-88 and N65A mutant genes, were also synthesized (GenScript) and all constructs were cloned into pLJ6, a mammalian expression vector derived from pHLsec3 (ref. ).

Techniques: Purification, Glycoproteomics, Functional Assay, Residue, Sequencing, Modification

Journal: Nature Structural & Molecular Biology

Article Title: Structure of the decoy module of human glycoprotein 2 and uromodulin and its interaction with bacterial adhesin FimH

doi: 10.1038/s41594-022-00729-3

Figure Lengend Snippet: a , The FimH-binding high-mannose glycan attached to UMOD N275 is located in the groove between the β-hairpin and D10C domain moieties of the protein’s decoy module (left panel). Although this sequon is not conserved in the decoy module of GP2, the groove of the latter contains a different, but closely spaced, N-glycosylation site at position 65 (right panel). b , SEC analysis of the material eluted after incubating an E. coli periplasmic extract containing untagged FimH L with wild-type or N65A mutant GP2 decoy modules immobilized on IMAC beads (left panels). Reducing SDS-PAGE analysis of the corresponding peak fractions (right panels) shows that FimH L binds to the wild-type GP2 decoy module but not to the N65A mutant. n = 2.

Article Snippet: A corresponding gene and an equivalent UMOD construct, as well as GP2 Δ31-59, Δ31-88 and N65A mutant genes, were also synthesized (GenScript) and all constructs were cloned into pLJ6, a mammalian expression vector derived from pHLsec3 (ref. ).

Techniques: Binding Assay, Glycoproteomics, Mutagenesis, SDS Page