Furin initiates gelsolin familial amyloidosis in the Golgi through a defect in Ca(2+) stabilization

Abstract

Hereditary familial amyloidosis of Finnish type (FAF) leading to amyloid in the peripheral and central nervous systems stems from deposition of a 71 residue fragment generated from the D187N/Y variants of plasma gelsolin by two sequential endoproteolytic events. We identify the protease accomplishing the first cleavage as furin, a proprotein convertase. Endoproteolysis of plasma gelsolin occurs in the trans-Golgi network due to the inability of the FAF variants to bind and be stabilized by Ca(2+). Secretion and processing of the FAF variants by furin can be uncoupled by blocking the convergence of the exocytic pathway transporting plasma gelsolin and the endocytic recycling of furin. We propose that coincidence of membrane trafficking pathways contributes to the development of proteolysis-initiated amyloid disease.

Fig. 1. Expression and processing of the wild-type and D187N FAF variant PGs. COS (A) or BHK (B) cells were transfected and radiolabeled with [³⁵S]methionine, and PG was immunoprecipitated from cells or medium at the indicated time as described in Materials and methods. Asterisks indicate low levels (<1%) of cross-reacting protein(s). The positions of the 83 kDa (full-length) and 68 kDa fragment (C68) are indicated by arrowheads. (C) Quantitation of data presented in (A) and (B). The results are typical of three independent experiments.

Fig. 2. Processing of D187N to C68 occurs in the Golgi. BHK cells were co-transfected with wild-type (ctl) or the D187N FAF variant (ctl, a–f) and the indicated Sar1, Arf1 or Rab GTPase mutants. Following expression, cells were washed, and the intracellular or secreted full-length C68 forms and recovered after a 2 h time period were analyzed by immunoblotting (A) and quantitated (B) as described in Materials and methods. Control (ctl) is the amount of wild-type or D187N secreted or processed in the absence of mutant GTPases. The results are typical of three independent experiments.

Fig. 3. α-gelsolinase is a furin-like proprotein convertase. (A) COS cells were transfected with either wild-type PG alone (a) and with furin (b) or the D187N variant alone (c) and with furin (d), labeled with [³⁵S]methionine, chased for the indicated time, and intracellular and secreted forms of PG immunoprecipitated. Asterisks indicate low levels (<1%) of cross-reacting protein(s). (B) Quantitation of data presented in (A). The results are typical of at least four independent experiments. (C) Expression of PG in the LoVo cell line lacking furin. Cells were mock transfected (a), transfected with D187N alone (b) or co-transfected with furin and D187N PG (c). The secreted forms reflecting the level of intracellular processing are shown. (D) Distribution of furin and PG in COS cells using indirect immunofluorescence as described in Materials and methods. Arrows indicate overlap in the TGN. Arrowheads denote furin-containing punctate endosomal compartments.

Fig. 3. α-gelsolinase is a furin-like proprotein convertase. (A) COS cells were transfected with either wild-type PG alone (a) and with furin (b) or the D187N variant alone (c) and with furin (d), labeled with [³⁵S]methionine, chased for the indicated time, and intracellular and secreted forms of PG immunoprecipitated. Asterisks indicate low levels (<1%) of cross-reacting protein(s). (B) Quantitation of data presented in (A). The results are typical of at least four independent experiments. (C) Expression of PG in the LoVo cell line lacking furin. Cells were mock transfected (a), transfected with D187N alone (b) or co-transfected with furin and D187N PG (c). The secreted forms reflecting the level of intracellular processing are shown. (D) Distribution of furin and PG in COS cells using indirect immunofluorescence as described in Materials and methods. Arrows indicate overlap in the TGN. Arrowheads denote furin-containing punctate endosomal compartments.

Fig. 4. α1-PDX inhibits D187N variant processing. (A) BHK cells were transfected with the D187N variant. Following 5 h of expression, the indicated amount of peptide inhibitor (dRVKRck) was added to the medium for 1 h, cells were washed, and the intracellular or secreted full-length and C68 forms recovered after a 2 h time period were analyzed by SDS–PAGE and immunoblotting. (B) Upper panel: BHK cells were co-transfected with the D187N variant and α1-PDX. Following 6 h of expression, cells were washed, and the intracellular or secreted full-length and C68 forms recovered after a 2 h time period were analyzed by immunoblotting. Lower panel: quantitation of data presented in the upper panel. Asterisks indicate low levels (<1%) of cross-reacting protein(s). The results are typical of two independent experiments.

Fig. 5. Furin-like PCs constitute the major pathway of FAF variant processing in different tissues. (A) BHK cells were co-transfected with the D187N variant and the indicated PCs. Following 6 h of expression, cells were washed, and the intracellular or secreted full-length and C68 forms recovered after a 2 h time period were analyzed by immunoblotting. (B) Quantitation of the data presented in (A).

Fig. 6. Furin endocytic trafficking pathways leading to processing of variant PG to C68. BHK cells were co-transfected with the wild-type (ctl) or the D187N FAF variant (ctl, a–c) and the indicated Rab GTPase mutants. Following 6 h of expression, cells were washed and the intracellular or secreted full-length and C68 forms recovered after a 2 h time period were analyzed by immunoblotting (A) and quantitated (B). The results are typical of two independent experiments.

Fig. 7. Ca²⁺ binding stabilizes wild-type PG but not the FAF variants. The stability of gelsolin towards thermal (A) and chaotrope (urea) denaturation (B) as described in Materials and methods in the presence and absence of Ca²⁺ reveals that wild-type gelsolin experiences a +11°C shift in T_m in the presence of 100 µM Ca²⁺, whereas the FAF variants display identical T_ms in both the presence and absence of Ca²⁺. The urea concentration required to half-denature the wild-type protein increases from 1.4 to 2.3 M in the presence of Ca²⁺, whereas the D187N/Y FAF variants display values that do not change significantly in the presence of Ca²⁺. See Table I for a summary of the thermo dynamic data. (C) The isothermal titration calorimetry binding isotherm resulting from the addition of 5 µl aliquots of 520 µM CaCl₂ to the wild-type gelsolin domain 2 construct (50 µM) is depicted (raw data presented as the insert). Analogous experiments with D187N and D187Y reveal no heat evolution and hence no binding of Ca²⁺.

Fig. 8. Variant processing is a consequence of the defect in Ca²⁺-mediated stabilization. The susceptibility of wild-type and the FAF variants (D187N/Y) of folded gelsolin domain 2 (134–266) (2 µM) to cleavage by soluble furin (2 U) under conditions likely to be similar to those found in the TGN (pH 6.2, 37°C, 1 mM CaCl₂). Proteolysis or lack thereof was evaluated as a function of time where the initial and 40 min time point are displayed in the SDS–polyacrylamide gel. Numerous experiments reveal that the wild-type domain is not susceptible to furin-mediated proteolysis whereas the FAF variants are proteolyzed into the expected 10.4 and 4.3 kDa fragments.