Abstract

Autosomal dominant Stargardt-like macular dystrophy (STGD3) in humans results from mutations in elongation of very long chain FAs-like 4 (ELOVL4), which leads to vision loss in young adults. ELOVL4 is an integral endoplasmic reticulum (ER) protein that mediates the elongation of very long chain (VLC) FAs. Mutations in ELOVL4 lead to truncation and mislocalization of the translated protein from the ER, the site of FA elongation. Little is known about the enzymatic elongation of VLC-FAs by ELOVL4. We over-expressed full-length mouse ELOVL4, an N-glycosylation-deficient mutant, an ER-retention mutant, and mutants of active site histidines to parse their individual roles in VLC-FA elongation. ELOVL4 elongated appropriate precursors to the corresponding VLC-FA species ≥28 carbons. Active site histidine mutants of ELOVL4 did not elongate appropriate precursors, establishing ELOVL4 as the elongase. Displacing ELOVL4 from the ER was sufficient to cause loss of condensation activity, while absence of N-glycosylation was irrelevant for enzyme function. This study shows that ELOVL4 enzymatic activity is governed by individual histidines in its active site and the ER microenvironment, both of which are essential for elongation of VLC-FAs.

Highlights

  • Autosomal dominant Stargardt-like macular dystrophy (STGD3) in humans results from mutations in elongation of very long chain FAs-like 4 (ELOVL4), which leads to vision loss in young adults

  • We recently showed that ELOVL4 mediated the elongation of 34:5n3 to 36:5n3 PUFA [9]; catalysis of successive steps from C28 to C34 by ELOVL4 has not been established

  • There is a low level of endogenous human ELOVL4 mRNA expression in the HEK293T cells, which was detected by quantitative real-time RT-PCR [9]

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Summary

Introduction

Autosomal dominant Stargardt-like macular dystrophy (STGD3) in humans results from mutations in elongation of very long chain FAs-like 4 (ELOVL4), which leads to vision loss in young adults. In this study we sought to characterize the enzymatic activity of the ELOVL4 protein and the contribution of each of the protein motifs toward the synthesis of VLC-FAs. We hypothesized that ELOVL4 is the elongase that mediates successive elongation steps generating FA chain lengths у28 carbons, and that these individual motifs are critical for enzyme function. ELOVL4-expressing cells elongated VLC-PUFA precursors 34:5n3 to 36:5n3 and 38:5n3, while controls showed no detectable levels of these elongated products (Fig. 1F).

Results
Conclusion

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