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Glycoproteomic Analysis Reveals New Pluripotency-associated Cell Surface Marker

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Review of "Leucine-Rich Repeat Neuronal Protein 1 Regulates Differentiation of Embryonic Stem Cells by Post-Translational Modifications of Pluripotency Factors" from STEM CELLS by Stuart P. Atkinson

The identification of specific surface markers for human embryonic stem cells (hESCs) will not only help us to understand the molecular mechanisms of stem cell differentiation and specific cell lineage production but can also contribute to ensuring the safety of hESC cell-derived grafts in clinical application [1]. Transcriptomics and proteomics have proven helpful techniques in the pursuit of novel markers; however, glycoproteomic analysis [2] may provide a better means to study hESC-specific cell surface markers, given the glycosylated nature of most secreted or cell surface proteins and the role of glycoconjugates in determining cell fate.

Now, a new STEM CELLS study from the laboratory of John Yu (Chang Gung Memorial Hospital at Linkou, Taoyuan, Taiwan) reports on a novel stem cell marker (LRRN1) discovered during the comparison of glycoprotein profiles of undifferentiated hESCs and embryoid body (EB) differentiated hESCs that plays a crucial role in proliferation, stemness, and differentiation [3]. Has glycoproteomic analysis uncovered a new pluripotency-associated cell surface marker?

Via glycoproteomic analysis (confirmed by subsequent studies of gene and protein expression), Liao et al. discovered the abundant expression of the neuronal leucine-rich repeat protein family member LRRN1 in hESCs; however, this waned upon EB-based differentiation suggesting an association of LRRN1 with the pluripotent state. Investigations into protein localization employed a specific monoclonal antibody generated by the laboratory, and demonstrated the presence of LRNN1 on the surface of hESCs, but not EB-differentiated cells.

Interestingly, upon LRRN1 silencing via short hairpin RNA expression, hESCs lost their ability to self-renew, displayed reduced protein levels of the OCT4, NANOG, and SOX2 pluripotency-associated factors, and differentiated preferentially to the endodermal and mesodermal lineages during in vitro assays and in vivo teratoma assays. However, the mRNA levels of the pluripotency factors did not alter in response to LRRN1 silencing, and the authors went on to discover that loss of LRRN1 led to the loss of AKT-mediated phosphorylation activity, the nuclear export of OCT4, NANOG, and SOX2 to the cytoplasm sue to a lack of phosphorylation [4, 5], and their proteasome-mediated degradation. The authors propose that LRRN1 activates AKT phosphorylation, although they did not address the question of direct or indirect regulation.

While studies have previously suggested a function for the little-known LRRN1 in cell adhesion and neural development [6, 7], this report represents the first description of a role for LRRN1 in the pluripotent nature of hESCs, thereby establishing LRRN1 as a potentially useful cell marker. The overall model suggests that LRRN1 helps to maintain the phosphorylation status of pluripotency-associated proteins, which in turn prevents their translocation from the nucleus to the cytoplasm, and thereby increases their stability and activity in hESCs.

For more on how glycoproteomic analysis can lead to the identification of new and useful cell surface markers, stay tuned to the Stem Cells Portal.

References

  1. Hentze H, Graichen R, and Colman A, Cell therapy and the safety of embryonic stem cell-derived grafts. Trends Biotechnol 2007;25:24-32.
  2. Hanson SR, Hsu TL, Weerapana E, et al., Tailored glycoproteomics and glycan site mapping using saccharide-selective bioorthogonal probes. J Am Chem Soc 2007;129:7266-7.
  3. Liao C-H, Wang Y-H, Chang W-W, et al., Leucine-Rich Repeat Neuronal Protein 1 Regulates Differentiation of Embryonic Stem Cells by Post-Translational Modifications of Pluripotency Factors. STEM CELLS 2018;36:1514-1524.
  4. Jeong CH, Cho YY, Kim MO, et al., Phosphorylation of Sox2 cooperates in reprogramming to pluripotent stem cells. Stem Cells 2010;28:2141-50.
  5. Lin Y, Yang Y, Li W, et al., Reciprocal regulation of Akt and Oct4 promotes the self-renewal and survival of embryonal carcinoma cells. Mol Cell 2012;48:627-40.
  6. Hayata T, Uochi T, and Asashima M, Molecular cloning of XNLRR-1, a Xenopus homolog of mouse neuronal leucine-rich repeat protein expressed in the developing Xenopus nervous system. Gene 1998;221:159-66.
  7. Buchanan SG and Gay NJ, Structural and functional diversity in the leucine-rich repeat family of proteins. Prog Biophys Mol Biol 1996;65:1-44.