Lysosome-associated membrane protein 1 is a major SSEA-1-carrier protein in mouse neural stem cells

Abstract

Stage-specific embryonic antigen-1 (SSEA-1) is a well-known carbohydrate antigenic epitope of undifferentiated cells, including neural stem cells (NSCs). However, the exact nature of the carrier proteins has not been fully characterized. Using proteomics analyses, we herein report that a lysosomal protein, LAMP-1, is a major carrier protein of SSEA-1 in NSCs, despite the common belief that SSEA-1 is mainly expressed on the cell surface and constitutes a component of the extracellular matrix. Furthermore, we found that SSEA-1 on LAMP-1 is completely ablated in differentiated cells derived from NSCs. Our finding raises the possibility that the expression of SSEA-1-positive LAMP-1 is associated with the "stemness" of NSCs.

Fig. 1

Detection of SSEA-1-carrier proteins in NSCs and differentiated cells. Proteins from primary (lane 1), secondary (lane 3) and tertiary (lane 5) neurospheres and cells differentiated from primary (lane 2), secondary (lane 4) and tertiary (lane 6) neurospheres were analyzed by western blot with AK97 anti-SSEA-1 antibody or anti-β-actin antibody (blot). β-Actin was detected as a loading control. Arrow indicates a major band positive for SSEA-1 with a molecular mass of 80 kDa. The bands indicated by asterisk were nonspecifically detected by the secondary antimouse IgM antibody.

Fig. 2

LAMP-1 as an SSEA-1-carrier protein. LAMP-1 was identified as a major SSEA-1-carrier protein by LC-MS/MS analysis after enrichment by immunoprecipitation with AK97 antibody. (A) Amino acid sequence of LAMP-1. The sequences of observed peptides are underlined. The putative signal sequence and transmembrane domain are marked by double and dashed underlines, respectively. Twenty potential N-glycosylation sites are indicated by asterisks. (B) Proteins from the tertiary neurospheres were immunoprecipitated with control mouse IgM or AK97 (IP) and then subjected to western blot analysis with anti-LAMP-1 antibody (left panel). The cell lysate was detected by AK97 as a control (right panel). (C) The lysates of tertiary neurospheres (containing 20 μg of proteins) were incubated with PNGase F (0 or 50 units/20 μL) at 37°C for 3 h to remove N-glycans and then analyzed by western blot with AK97, anti-LAMP-1 antibody and anti-β-actin antibody.

Fig. 3

Localization of SSEA-1 in NSCs. NSCs prepared from neurospheres were treated with PBS containing 3% fetal bovine serum and 0% or 0.1% Triton X-100 (Trx100) and then stained with AK97 and Alexa Fluor 488-conjugated antimouse IgM antibody (green). Nuclei were stained with 2 μg/mL of Hoechst 33258 (H33258; blue).

Fig. 4

LAMP-1 expressed in NSCs and differentiated cells. (A) Cell lysates from primary (lane 1), secondary (lane 3) and tertiary (lane 5) neurospheres and cells differentiated from primary (lane 2), secondary (lane 4) and tertiary (lane 6) neurospheres were analyzed by western blot with anti-LAMP-1 antibody or anti-β-actin antibody. (B) The mRNA expression of LAMP-1, FUT4, FUT9, FUT10, FUT11, Sox2 and β-actin in undifferentiated NSCs (undiffer) and differentiated cells (differ) were analyzed by RT-PCR. Sox2 was detected as a marker gene of undifferentiated NSCs. β-Actin was used as a control.