Repeat domain-associated O-glycans govern PMEL fibrillar sheet architecture

Morven Graham,Ralf M Leonhardt,Susan M Mitchell,Xinran Liu,Athanasia C Tzika

doi:10.1038/s41598-019-42571-6

Abstract

PMEL is a pigment cell-specific protein that forms a functional amyloid matrix in melanosomes. The matrix consists of well-separated fibrillar sheets on which the pigment melanin is deposited. Using electron tomography, we demonstrate that this sheet architecture is governed by the PMEL repeat (RPT) domain, which associates with the amyloid as an accessory proteolytic fragment. Thus, the RPT domain is dispensable for amyloid formation as such but shapes the morphology of the matrix, probably in order to maximize the surface area available for pigment adsorption. Although the primary amino acid sequence of the RPT domain differs vastly among various vertebrates, we show that it is a functionally conserved, interchangeable module. RPT domains of all species are predicted to be very highly O-glycosylated, which is likely the common defining feature of this domain. O-glycosylation is indeed essential for RPT domain function and the establishment of the PMEL sheet architecture. Thus, O-glycosylation, not amino acid sequence, appears to be the major factor governing the characteristic PMEL amyloid morphology.

Highlights

PMEL forms a functional amyloid matrix in melanosomes of pigment cells[1,2] and is a potent melanoma antigen[3,4]
We demonstrate that the RPT domain, despite differing dramatically in amino acid sequence between species, is a functional module, which is largely interchangeable between PMEL orthologues
The RPT domain is required for normal melanosomal amyloid morphology

Summary

Introduction

PMEL forms a functional amyloid matrix in melanosomes of pigment cells[1,2] and is a potent melanoma antigen[3,4]. The amyloid core of the fibrils is formed by a proteolytic product derived from the PMEL protein[1]. This proteolytic fragment assembling into and forming the core was originally discovered via its reactivity with antibody I517. There is a second type of proteolytic fragment associated with melanosomal fibrils, MαC9, which is derived from the PMEL protein and which contains the polycystic kidney disease (PKD) domain and the highly O-glycosylated repeat (RPT) domain[8,9]. Precisely how the amyloid structure is altered in the absence of the repeat domain is difficult to understand from electron microscopy (EM) two-dimensional imaging This is an important problem, because the shape of the matrix is tightly linked to its function, as its organization into sheets maximizes the surface area available for melanin deposition. We demonstrate that the RPT domain, despite differing dramatically in amino acid sequence between species, is a functional module, which is largely interchangeable between PMEL orthologues

Methods

Results

Conclusion