Research Article

Tyrosinase processing and intracellular traf-

ficking is disrupted in mouse primary melanocytes

carrying the underwhite (uw) mutation. A model for oculocutaneous albinism (OCA)

type 4 2013 l 08 l 03 PAPER STUDY

By Eun-Kyung

OJECTIVETo analyze the intracellular processing and trafficking of melanogenic proteins in primary uw-mutant melanocyte as a model for OCA type 4, focusing on tyrosi-nase.

SUMMARY

Oculocutaneous albinism (OCA) type 4 is a newly identified human autosomal recessive hypopigmentary disorder that disrupts pigmentation in the skin, hair and eyes. Three other forms of OCA have been previously characterized, each resulting from the aberrant processing and/or sorting of tyrosinase, the enzyme critical to pigment production in mammals. The disruption of tyrosi-nase trafficking occurs at the level of the endoplasmic reticulum (ER) in OCA1 and OCA3, but at the post-Golgi level in OCA2. The gene responsible for OCA4 is the human homologue of the mouse underwhite (uw) gene, which encodes the membraneassociated transporter protein (MATP). To characterize OCA4, we investigated the processing and sorting of melanogenic proteins in primary melanocytes derived from uw/uw mice and from wild-type mice. OCA4 melanocytes were found to be constantly secreted into the medium dark vesicles that contain tyrosinase and two other melanogenic enzymes, Tyrp1 (tyrosinase-related protein 1) and Dct (DOPAchrome tautomerase); this secretory process is not seen in wild-type melanocytes. Although tyrosinase was synthesized at comparable rates in wild-type and in uwmutant melanocytes, tyrosinase activity in uw-mutant melanocytes was only about 20% of that found in wild-type melanocytes, and was enriched only about threefold in melanosomes compared with the ninefold enrich-ment in wild-type melanocytes. OCA4 melanocytes showed a marked difference from wild-type melanocytes in that tyrosinase was abnormally secreted from the cells, a process similar to that seen in OCA2 melanocytes, which results from a mutation of the pink-eyed dilution (P) gene. The P protein and MATP have 12 transmembrane regions and are predicted to function as transporters. Ul-trastructural analysis shows that the vesicles secreted from OCA4 melanocytes are mostly early stage melanosomes. Taken to-gether, our results show that in OCA4 melanocytes, tyrosinase processing and intracellular trafficking to the melanosome is dis-rupted and the enzyme is abnormally secreted from the cells in immature melanosomes, which disrupts the normal maturation process of those organelles. This mechanism explains the hypopigmentary phenotype of these cells and provides new insights into the involvement of transporters in the normal physiology of melanocytes.

Introduction

Material and Methods

Results

Discussion

ALBINISMHetero group of genetic hypopigmentary disorders

OA (Ocular Albinism) : pigmentation in the visual system, mild formOCA (Oculocutaneous Albinism) : reduced/absent pigmenata-tion

OCA1 Tyrosinase• Autosomal recessive disorder associated with deficient cat-alytic function of tyrosinase• Endoplasmic reticulum retention disease

OCA2 P protein• Most common albinism, minimal-to-moderate pigmentation• Correctly processed through Golgi, but secreted from the cells• Altered intracellular trafficking of tyrosinase

OCA3 Tyrp1• Rare form• Same with OCA1(Endoplasmic reticulum retention disease)

OCA4 MATP• New distinct type

MATP ?

Membrane-associated transporter pro-

tein

Mitf - transcriptional level

12 predicted membrane-spanning re-

gion

Introduction

Material and Methods

Results

Discussion

Cell cul-ture

Antibodies and En-zymes

Celullar fractiona-tion

Metabolic label-ing

Western blot-ting

EndoH and PNGase diges-tions

Melanogenic as-says

Immunohistochemical staining

Electron mi-croscopy

Primary melanocyte from wild-type, uw-mutant, albino C57BL/6J

αPEP1, αPEP7, αPEP8, anti-rabbit IgG horseradish peroxidase-linked, KDEL, HMB-45, EndoH, PNGaseF etc

Extraction buffer,- Vesicles secreted by melanosome- Melanosomal –enriched fraction

6 well tissue culture0.5mCi of[35S]-Methionine and Cys-teine mixture, αPEP1, αPEP7, 8% SDS-PAGE

8% SDS-PAGE, PVDF, first antibody 1:1000, anti-rabbit IgG horseradish peroxidase-linked whole antibody 1:1500

Samples to be digested

Tyrosinase enzymatic activity, melanin contents

Dual labeling; immunofluorescence method and laser scanning confocal fluorescence microscopy( green, red, yellow)

2% glutaraldehide and 1% osmium tetroxide fixation, embedded in PolyBed 812, ultrathin section, uranyl acetate stainingJEOL JEM-1200 EX electron microscopy

Introduction

Material and Methods

Results

Discussion

Figure 1 Characteristics of wild-type and uw-mutant primary murine melanocytes in culture

• The growth of two type were quite similar

• Morphologies quite stable

• Uw-mutant melanocyte : larger, more dendritic

• Wild type - very pigmented population (C) - no dark vesicle (E)

• un-mutant - very lightly pigmented(D) - dark vesicle detected (F) into the medium

Introduction

Material and Methods

Results

Discussion

Figure 1 Characteristics of wild-type and uw-mutant primary murine melanocytes in culture

Introduction

Material and Methods

Results

Discussion

(A,B) Phase contrast photomicrographs of wild-type and uw-mutant melanocytes.

(C,D) Bright-field microphotographs emphasizing the pigmentation of wild-type

and uw-mutant melanocytes.

(E,F) Bright-field microphotographs showing the pigmented vesicles secreted by uw-mu-

tant

melanocytes into the medium (arrows) and their absence in the medium of wild-type

melanocytes. All photomicrographs are shown at the same magnification.

(G) Media were collected from wild-type and from uw-mutant melanocytes and were cen-

trifuged

to show the similar appearance of those samples (i.e. no soluble melanin was present).

(H) Pigmented vesicles released into the medium by uw-mutant melanocytes can be read-

ily

sedimented by centrifugation, a process not seen in wild-type melanocytes.

(I) Melanin content of extracts of wild-type and uw-mutant melanocytes.

X 9

Below 1%

X 3

Over 5%

Introduction

Material and Methods

Results

Discussion

Figure 2 Metabolic pulse-chase labeling of wild-type and uw-mutant melanocytes. Wildtype, uw-mutant and Tyrc albino primary melanocytes were labeled for 1 hour with an [35S]-me-thionine and cysteine mixture, harvested at the chase times indicated, solubilized and im-munoprecipitated using specific antibodies for tyrosinase and Tyrp1, respectively.

√ To examine the synthesis and processing of tyrosinase and Tyrp1 as each

melanocyte

√ Stability of tyrosinase : wild type-100% remaining, uw-mutant-very little after

24hr

Fully glycosylatiedEarly glycosylation

Start polypeptide

Introduction

Material and Methods

Results

Discussion

Figure 3 Western blot analysis of melanogenic protein glycosylation in subcellular fractions of wild-type and of uw-mutant melanocytes. Glycosylation of melanosomal proteins was determined following di-gestion with buffer (–), EndoH (E) or PNGaseF (P), as indicated.

Fully glycosylatiedEarly glycosylation

Start polypeptide

√ Tyrosinase of wild type : none detection of secreted vesicle

√ Tyrp1 processed correctly and found in melanosome of uw-mutant

and also present secreted vesicle fraction

Introduction

Material and Methods

Results

Discussion

Figure 4 Subcellular distribution of melanogenic proteins in wild-type and uw-mutant primary melanocytes.

Marker for the ER

Marker for the early (stage Ⅱ) melanosome

√ To investigate the subcellular localization of melanogenic protein in uw-mutant OCA4

√ In wild-type : tyrosinase-perinuclear area(ER, stageⅠ melanosome), little co-staining with

HMB-45

√ In uw-mutant : more dendritic and much larger size

• Tyrosianse, Tyrp1, Dct : Red

• KDEL, HMB-45 : green

• Colocalization of Ab : yellow

Introduction

Material and Methods

Results

Discussion

Figure 5 Ultrastructure of primary melanocytes established from wild-type and from uw-mutant mice.

(A) Wild-type melanocytes : numerous stage III and IV

melanosomes

(B) uw-mutant melanocytes : primarily stage I and II melanosomes

swollen, poorly melanized melanosomes

(C) uw-mutant melanocytes : DOPA-reactivity > positive dark stain-

ing

in stage I and II

revealed the presence of active tyrosinase

(D) Secreted vesicle fraction from uw-mutant melanocytes where

stage I and II melanosomes, similar to those found in (B)

Bar, 20 mm

Introduction

Material and Methods

Results

Discussion

Primary melanocyte so far available

OCA4 new type of oculocutaneous albinism mutation of underwhite (MATP) gene

Uw-mutant cells Expression normal level of tyrosinase Secretion of dark vesicle into medium > low melanin, 20% reduced tyrosinaes ac-tivity

Sorting of tyrosinase from trans-Golgi network to stage Ⅱ melanosome

Function of MATP(uw-encoded pro-tein)

Melanosome-specific protein but absence at stage Ⅱ melanosome > transport proetein

Similarity of OCA2/P protein phenotype, function

What is

this pa-

per

saying ?

Introduction

Material and Methods

Results

Discussion

Figure 6 Melanosomal protein trafficking and abberrant processing seen in various of OCA.

√ Various type of OCA

√ OCA1, 3 : disruption of tyrosinase trafficking at the level of ER

√ OCA2 : disruption of tyrosinase trafficking at the post-Golgi

level

√ OCA4 : cell secretion within vesicles before delivery