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    • Vesicular Transport and Polarity: Cargo Receptors

    Klaus Fiedler

    Recent projects in my career centered on epithelial polarity (1990-1995). The exocytic carrier vesicles (TGN-derived) of polarized epithelial cells were analyzed (see above) at the EMBL (Prof. Kai Simons` laboratory) and led to the novel finding of a 36 kD transmembrane protein with a homology to leguminous plant lectins . This discovery paved the way for lectin and carbohydrate studies on a family of plant lectin-similar proteins which today are known to regulate and stimulate transport in the secretory pathway, VIP36 interacted with N-glycans in a reasonably small library of glycans (see A) and showed a small pH-dependence in relation to other mammalian legume-type lectins. Moreover, my own studies showed that the interaction with carbohydrates on cells could be abolished with monosaccharides, which indicates that glycolipids or O-glycans containing GalNAc may contribute to its carbohydrate binding specificity (B), and furthermore could suggest a role of VIP36 in protein targeting in renal and intestinal epithelial cells . It was previously shown, that apical secretion of N-glycosylated secretory proteins involved VIP36 expression and was reduced in VIP36 mutant MDCK (Madin-Darby Canine Kidney) cells . The result obtained by the docking analysis suggests, that the binding of O-glycans to VIP36 may, however, show the highest differential energy in interaction depending on calcium availability in the calcium binding-site alteration in the Asp131-Asn Asn166-Asp change of VIP36 (C) (unpublished). It seems, that a role of VIP36 in interaction and transport of O-glycosylated proteins has so far remained enigmatic.
    Some lectins in the secretory pathway and Golgi apparatus are shown in panel D. VIP36 docking data are summarized in panel E introducing 3 different conformers to the docking algorithm: Top scoring carbohydrates are of high-mannose type and a fucoside, on average, high-mannose N-glycans are topped by complex N-glycans. Interestingly, differences of VIP36 and ERGIC53, a paralogue, in glycan affinity are revealed in a comparison using an identical database of glycans for molecular docking (F).
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    A  VIP36 and N-Glycan Interaction

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    B  Putative O-Glycan Interaction

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    C  Asp-Asn Asn-Asp VIP36-Mutational Docking

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    D  Lectins in the Secretory Pathway

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    E  Summary of Docking Data VIP36

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    F  Glycan Binding: VIP36 relative to ERGIC53

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    G  Result on N-Glycosylation of VIP36

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    H  Annexin Model: AnxA13b

    Recent work (2017) shows that VIP36 (LMAN2) was N-glycosylated in embryonic stem cells and contains a sialylated complex N-glycan (G) . In addition to aforementioned novel data on VIP36 it has also been found, that the α1,6-fucosyltransferase 8 (FUT8) knockout in CHO-cells, in an unbiased screen, strongly correlated with VIP36 expression , lending credence to the model obtained from molecular docking experiments on increased VIP36 binding to core-fucosylated N-glycans . In the previous SARS-CoV-2 infection and interactome data (2020), it has surprisingly been demonstrated, that VIP36 is one component of several trafficking-associated factors that is shown to interact with nsp7 , a non-structural protein of the coronavirus. Why the replication machinery of the coronavirus (nsp7 is bound to nsp8 and to nsp12 polymerase) attaches to the VIP36 is molecularly not understood but may target the virus core or surface proteins to the site of viral genesis and formation in the secretory pathway (see a recent confirmation including interactome data ).
    A complex has been described (2019), which includes abundant TMED proteins (TMED2, -4, -5 -7, -9, -10), VIP36, GRASP55 and GM130 that associated with misfolded prion protein (PrP*) on its way through the Golgi apparatus to the plasma membrane from where it is destined to lysosomes . This suggests that supramolecular protein complexes of cargo receptors may carry out functions as chaperones of factors on their way through the Golgi apparatus as well. Interestingly, both TMED proteins and VIP36 had previously been found to interact with HIV proteins as analyzed by mass-spectrometry: TMED4, -9 and -10 were interacting with the Rev protein, VIP36 was found to interact with Gp160 and Gp41 , which had only been independently confirmed for the latter interactions (RNAi screen/virus replication) .

    Annexin A13

    In a separate work, I demonstrated that annexin A13b was involved in apical exocytic transport . This showed for the first time, that apical delivery in polarized MDCK cells implicates annexins and further may entail SNARE proteins . A structure of annexin A13 (AnxA13) from rat and a model of a newly aligned dog AnxA13 suggest, that annexins may unfold to act as membrane integral ion channels, a proposal built on annexin B12 and annexin A5 biophysical analyses (H). Thus, regulation of the lipid membrane inner fluidity-gradient just as the formation of a protein-lined ion channel within the membrane, may be one of the roles played by annexin proteins.

    VIP17

    In a further collaboration, VIP17-MAL, previously described as a protein common to apical and basolateral transport vesicles in MDCK cells, was microsequenced. By confocal microscopy the protein could be identified in punctate vesicular structures and the plasma membrane with a preference for the apical pole in comparison to VIP21-caveolin . The MAL family of proteins displays a very high hydrophobicity with multiple membrane-penetrating regions and interestingly, MALL, a further member of the protein family, was recently shown in PML (pro-myelocytic leukemia) nuclear bodies in A431 cells . Although likely different in function, the nuclear localization reminds of the SVEC4-10 (transformed cells) nuclear localization of caveolin-1 that we had previously found . This other major protein of epithelial trans-Golgi network-derived transport vesicles, VIP21-caveolin-1, was identified in 1992 by Kurzchalia et al. and is today described as a possible semi-membrane penetrating ion- or lipid-channel that is awaiting further detailed description .
     
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    References
    1. Fiedler K, Parton RG, Kellner R, Etzold T, Simons K. VIP36, a novel component of glycolipid rafts and exocytic carrier vesicles in epithelial cells. EMBO J. 1994;13:1729-40.
    2. Fiedler K. Vesicular traffic in polarized epithelial cells: Identification and characterization of general and epithelial specific factors. [Basel]: Ph.D. Thesis. University of Basel; 1994.
    3. Kamiya Y, Yamaguchi Y, Takahashi N, Arata Y, Kasai K, Ihara Y, et al. Sugar-binding properties of VIP36, an intracellular animal lectin operating as a cargo receptor. J Biol Chem. 2005;280(44):37178–37182.
    4. Fiedler K, Simons K. Characterization of VIP36, an animal lectin homologous to leguminous lectins. J Cell Sci. 1996;109(Part 1):271-6.
    5. Fiedler K, Simons K. The role of N-glycans in the secretory pathway. Cell. 1995;81:309-12.
    6. Hara-Kuge S, Ohkura T, Ideo H, Shimada O, Atsumi S, Yamashita K. Involvement of VIP36 in intracellular transport and secretion of glycoproteins in polarized Madin-Darby Canine Kidney (MDCK) cells. J Biol Chem. 2002;277(18):16332–16339.
    7. Stadlmann J, Taubenschmid J, Wenzel D, Gattinger A, Dürnberger G, Dusberger F, et al. Comparative glycoproteomics of stem cells identifies new players in ricin toxicity. Nature. 2017;549(7673):538–542.
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    10. Fiedler K. VIP36 preferentially binds to core-fucosylated N-glycans: a molecular docking study. bioRxiv 092460: doi: https://doi.org/10.1101/092460
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    16. Fiedler K, Lafont F, Parton RG, Simons K. Annexin XIIIb: a novel epithelial specific annexin is implicated in vesicular traffic to the apical plasma membrane. J Cell Biol. 1995;128(6):1043-1053.
    17. Lecat S, Verkade P, Thiele C, Fiedler K, Simons K, Lafont F. Different properties of two isoforms of annexin XIII in MDCK cells. J Cell Sci. 2000;113(Pt 14):2607–2618.
    18. Plant PJ, Lafont F, Lecat S, Verkade P, Simons K, Rotin D. Apical membrane targeting of Nedd4 is mediated by an association of its C2 domain with annexin XIIIb. J Cell Biol. 2000;149(7):1473–1483.
    19. Wang P, Chintagari NR, Gou D, Su L, Liu L. Physical and functional interactions of SNAP-23 with annexin A2. Am J Respir Cell Mol Biol. 2007;37(4):467-76.
    20. Umbrecht-Jenck E, Demais V, Calco V, Bailly Y, Bader M-F, Chasserot-Golaz S. S100A10-mediated translocation of annexin-A2 to SNARE proteins in adrenergic chromaffin cells undergoing exocytosis. Traffic. 2010;11(7):958-71.
    21. Gerke V, Creutz CE, Moss SE. Annexins: Linking Ca2+ signalling to membrane dynamics. Nat Rev Mol Cell Biol. 2005;6:449–461.
    22. Zacchetti D, Peranen J, Murata M, Fiedler K, Simons K. VIP17/MAL, a proteolipid in apical transport vesicles. FEBS Lett. 1995;377(3):465–469.
    23. Rubio-Ramos A, Bernabé-Rubio M, Labat-de-Hoz L, Casares-Arias J, Kremer L, Correas I, et al. MALL, a membrane-tetra-spanning proteolipid overexpressed in cancer, is present in membraneless nuclear biomolecular condensates. Cell Mol Life Sci. 2022 Apr;79(5):236.
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    25. Kurzchalia T V, Dupree P, Parton RG, Kellner R, Virta H, Lehnert M, et al. VIP21, a 21-kD membrane protein is an integral component of trans-Golgi-network-derived transport vesicles. J Cell Biol. 1992;118:1003–1014.
    26. Porta JC, Han B, Gulsevin A, Chung JM, Peskova Y, Connolly S, et al. Molecular architecture of the human caveolin-1 complex. Sci Adv. 2022 Jan 30;8(19):eabn7232.

    Update 2022