LAMP-2C Inhibits MHC Class II Presentation of Cytoplasmic Antigens by Disrupting Chaperone-Mediated Autophagy

Abstract

Cells utilize multiple autophagy pathways to sequester macromolecules, senescent organelles, and pathogens. Several conserved isoforms of the lysosome-associated membrane protein (LAMP)-2 regulate these pathways influencing immune recognition and responses. LAMP-2A is required for chaperone-mediated autophagy (CMA) which promotes Ag capture and MHC class II (MHCII) presentation in B cells and signaling in T cells. LAMP-2B regulates lysosome maturation to impact macroautophagy (MA) and phagocytosis. Yet, far less is known about LAMP-2C function. While LAMP2A and LAMP2B mRNA were broadly detected in human tissues, LAMP2C expression was more limited. Transcripts for the three LAMP2 isoforms increased with B cell activation, although specific gene induction varied depending on TLR versus BCR engagement. To examine LAMP-2C function in human B cells and specifically its role in Ag presentation, ectopic gene expression was used. Increased LAMP-2C expression in B cells did not alter MHCII expression or invariant chain processing, but did perturb cytoplasmic Ag presentation via CMA. MHCII presentation of epitopes from exogenous and membrane Ags was not affected by LAMP-2C expression in B cells. Similarly, changes in B cell LAMP-2C expression did not impact MA. The gene expression of other LAMP2 isoforms as well as the proteasome and lysosomal proteases activities were unperturbed by LAMP-2C ectopic expression. LAMP-2C levels modulated the steady-state expression of several cytoplasmic proteins which are targeted for degradation by CMA and diminished peptide translocation via this pathway. Thus, LAMP-2C serves as a natural inhibitor of CMA which can selectively skew MHCII presentation of cytoplasmic Ags.

Transcription factor NFE2L2/NRF2 modulates chaperone-mediated autophagy through the regulation of LAMP2A

“…Although we could not detect differences in the expression levels of LAMP2B or LAMP2C upon genetic manipulation of NFE2L2 in human HEK293T cells, mouse hepatocytes or HT22 cells, reduced levels of LAMP2B mRNA were found in A549 cells upon NFE2L2 silencing, and NFE2L2-deficient MEFs displayed reduced levels of the 3 isoforms. Differential regulation of the 3 isoforms has been reported previously [10,15,42]. For example, paraquat transcriptionally induces Lamp2a expression without affecting the other 2 isoforms [43].…”

“…In fact, several roles have been proposed for LAMP2B and LAMP2C [10,38–40], but LAMP2A is the only isoform required for CMA [4,8,25]. LAMP2 isoforms display tissue-specific expression patterns [10,41].…”

“…LAMP2 isoforms display tissue-specific expression patterns [10,41]. While LAMP2A and LAMP2B are highly expressed in most tissues, LAMP2C appears to be very restricted [10], pointing to a different splicing regulation of the LAMP2 gene. Although we could not detect differences in the expression levels of LAMP2B or LAMP2C upon genetic manipulation of NFE2L2 in human HEK293T cells, mouse hepatocytes or HT22 cells, reduced levels of LAMP2B mRNA were found in A549 cells upon NFE2L2 silencing, and NFE2L2-deficient MEFs displayed reduced levels of the 3 isoforms.…”

“…All LAMP2A, LAMP2B and LAMP2C isoforms share an identical lumenal region but differ in their cytosolic and transmembrane tail [9]. These isoforms result from cell-specific yet unknown regulation of differential splicing [10,11] but experimental evidence supports the conclusion that LAMP2A is the only LAMP2 variant required for CMA [4,8]. …”

Melanoma LAMP-2C Modulates Tumor Growth and Autophagy

“…Primers for LAMP2 and GAPDH amplification were described (Perez et al, 2016). LAMP2 cDNA was amplified using 2X ReddyMix PCR Master Mix (Thermo Fisher Scientific, Waltham, MA, United States) for 35 cycles.…”