Synthetic glycopolymers that emulate cell-surface mucins have been used to elucidate the role of mucin overexpression in cancer. However, because they are internalized within hours, these glycopolymers could not be used to probe processes that occur on longer time scales. Here, we tested a panel of glycopolymers bearing a variety of lipids to identify those that persist on cell membranes. Strikingly, we found that cholesterylamine (CholA)-anchored glycopolymers are internalized into vesicles that serve as depots for delivery back to the cell surface, allowing for the display of cell-surface glycopolymers for at least 10 days, even while cells are dividing. As with native mucins, cell-surface display of CholA-anchored glycopolymers influenced focal adhesion distribution. Furthermore, we show that these mimetics enhance survival of nonmalignant cells in a zebrafish model of metastasis. CholA-anchored glycopolymers therefore expand applications of glycocalyx engineering in glycobiology.

Synthetic glycopolymers that emulate cell-surface mucins have been used to elucidate the role of mucin overexpression in cancer. However, because they are internalized within hours, these glycopolymers could not be used to probe processes that occur on longer time scales. Here, we tested a panel of glycopolymers bearing a variety of lipids to identify those that persist on cell membranes. Strikingly, we found that cholesterylamine (CholA)-anchored glycopolymers are internalized into vesicles that serve as depots for delivery back to the cell surface, allowing for the display of cell-surface glycopolymers for at least 10 days, even while cells are dividing. As with native mucins, cell-surface display of CholA-anchored glycopolymers influenced focal adhesion distribution. Furthermore, we show that these mimetics enhance survival of nonmalignant cells in a zebrafish model of metastasis. CholA-anchored glycopolymers therefore expand applications of glycocalyx engineering in glycobiology. KeywordsGlycopolymers; Glycoconjugates; Lipids; Cancer; Cell Adhesion Global changes in glycosylation can accompany inflammation, microbial infection and tumorigenesis. [1] For example, heavily glycosylated mucin glycoproteins have long been known to be upregulated in epithelial carcinomas, though until recently their contribution to oncogenesis remained largely mysterious. [2] Mucins are membrane-associated glycoproteins with densely glycosylated ectodomains; their glycans often comprise >50% of the mass of the molecule. [3] It was recently shown that mucins' biophysical bulk may actually bolster adhesion of tumor cells in minimal adhesion settings, such as the metastatic niche. [4] Additionally, broad upregulation of certain glycan motifs, such as sialic acid, can play an important role in how the tumor and the host immune system interact. [5] These and other studies of glycobiology have benefitted from chemical approaches, such as the display of synthetic glycoconjugates on live cells via chemical glycocalyx engineering. [6] One of the most straightforward methods to control cell surface glycan Correspondence to: Carolyn R. Bertozzi. HHS Public Access Author ManuscriptAuthor Manuscript Author ManuscriptAuthor Manuscript display is through passive insertion of lipid-anchored glycopolymers into the plasma membrane. [5,[7][8][9][10][11] This strategy offers many advantages over other techniques, such as covalent attachment of glycoconjugates to engineered cell-surface molecules. [11,12] Because lipid-anchored glycopolymers will insert into any plasma membrane, any naïve cell type can be used, including primary cells and those not amenable to genetic manipulation. However, previously reported lipid-conjugated glycopolymers, including those functionalized with dipalmitoyl phosphatidylethanolamine-based anchors (DPPE, 1, Fig. 1a), are constitutively internalized by cells. [8] As a consequence, their plasma membrane residence half-lives are only 5-6 hours, which limits their application to studies of biological processes occurr...

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The type I keratin 14 (K14) is mainly expressed in the basal layer of epidermis and related stratified epithelia. Previous work revealed a role for inter-keratin disulfide bonding involving residue cysteine (Cys) 367 of human K14 toward the assembly, organization, and dynamics of keratin filaments in skin keratinocytes in culture. To define the function of K14-dependent disulfide bonding in skin epithelia in vivo, we generated Krt14C373A mice using the Crispr/ Cas9 technology (mouse Cys373 is orthologous to human Cys367). Western blotting revealed a marked decrease in disulfide-bonded K14 species in Krt14C373A ear and tail skin relative to control. Morphological analyses revealed a state of hyperproliferation and hyperkeratosis, dysregulated keratinocyte differentiation, and abnormal cornified envelopes in Krt14C373A skin. Adult Krt14C373A mice show a skin barrier defect at baseline, reflected by increased trans-epidermal water loss. The latter trait is enhanced following topical acetone irritation. Mass spectrometry-based assays identified 14-3-3 as a major K14 interacting protein, and follow-up studies confirmed that 14-3-3 interacts with both WT and C373A mutant forms. 14-3-3 forms aggregates in Krt14C373A keratinocytes in situ, accompanied by abnormal nuclear localization of Yap1, a transcriptional effector of Hippo signaling, in suprabasal differentiated keratinocytes. Mutagenesis shows that the subcellular distribution and regulation of 14-3-3 and Yap1 is specifically regulated by residue Cys373 in mouse K14. The results obtained to date establish that K14-dependent disulfide bonding plays a physiologically important role in epidermal homeostasis and barrier function by impacting the solubility, regulation and function of 14-3-3/Yap1.