Helical stability of the GnTV transmembrane domain impacts on SPPL3 dependent cleavage

Abstract

Signal-Peptide Peptidase Like-3 (SPPL3) is an intramembrane cleaving aspartyl protease that causes secretion of extracellular domains from type-II transmembrane proteins. Numerous Golgi-localized glycosidases and glucosyltransferases have been identified as physiological SPPL3 substrates. By SPPL3 dependent processing, glycan-transferring enzymes are deactivated inside the cell, as their active site-containing domain is cleaved and secreted. Thus, SPPL3 impacts on glycan patterns of many cellular and secreted proteins and can regulate protein glycosylation. However, the characteristics that make a substrate a favourable candidate for SPPL3-dependent cleavage remain unknown. To gain insights into substrate requirements, we investigated the function of a GxxxG motif located in the transmembrane domain of N-acetylglucosaminyltransferase V (GnTV), a well-known SPPL3 substrate. SPPL3-dependent secretion of the substrate’s ectodomain was affected by mutations disrupting the GxxxG motif. Using deuterium/hydrogen exchange and NMR spectroscopy, we studied the effect of these mutations on the helix flexibility of the GnTV transmembrane domain and observed that increased flexibility facilitates SPPL3-dependent shedding and vice versa. This study provides first insights into the characteristics of SPPL3 substrates, combining molecular biology, biochemistry, and biophysical techniques and its results will provide the basis for better understanding the characteristics of SPPL3 substrates with implications for the substrates of other intramembrane proteases.

Cooperation of N- and C-terminal substrate transmembrane domain segments in intramembrane proteolysis by γ-secretase

“…For DHX analysis, we used synthetic peptides where the hydrophobic TMD residues are flanked by Lys triplets (Supplementary Table 1 ). Similar to our previous analysis of various substrate TMD peptides 8 – 11 , 27 – 29 , DHX kinetics of exhaustively (>95%) deuterated peptides were measured at 20 °C and pH 5 in 80% trifluoroethanol (TFE). The polarity of TFE roughly matches that within the solvated interior of a protein 30 and is therefore thought to mimic the aqueous environment within presenilin 31 .…”

“…Replacing A 42 G 43 A 44 by Leu stabilized the helix and strongly decreased cleavage at a downstream site of the TMD 45 . Likewisely, increased or decreased TMD helix flexibility facilitated or impeded SPPL3‑dependent shedding, respectively, of its substrate GnTV 29 .…”

Structure and function of SPP/SPPL proteases: insights from biochemical evidence and predictive modeling

“…Mutations at the proposed hinge in TNFα from AGA to helix stabilizing LLL reduced initial cleavage by SPPL2a [101]. In line with this, a proline mutation in a proposed hinge region in the TM domain of GNT‐V, a substrate of SPPL3, resulted in increased initial cleavage of the substrate [55]. Computational analysis of 23 SPPL3 substrates indicates a modest enrichment of glycine residues in the middle of the cleavable TM domain [118].…”

“…Experimental structural data as proof are however missing. Sequence analysis of cleavage regions so far has not led to the identification of consensus recognition sequences within the substrate's TM domains [2,55,117]. For SPPL3, it was suggested that an M or Y in position P1 might be favorable [33]; however, a recent N‐terminomics study on the enzyme did not detect a consensus sequence [118].…”

“…Mostly these substrate's TM domains have been released from the fulllength protein by an independent proteolytic cleavage in a neighboring loop domain ( [53,54]. SPPL3 releases the catalytic domain of a variety of type II glycosyltransferases to the Golgi lumen from where they are secreted, thus deactivating their function in the Golgi [22,31,55]. Whether this is a part of a signaling process or a degradation/downregulation mechanism has been extensively discussed and remains to be unraveled [42].…”