PIGMENT CELL RES 14: 243–248. 2001 Copyright © Pigment Cell Res 2001ISSN 0893-5785Printed in Ireland—all rights reser�ed

Review: Pigment Gene Focus

Ocular Albinism Type 1: More Than Meets The Eye

BIN SHEN, PREMINDA SAMARAWEERA, BENJAMIN ROSENBERG and SETH J. ORLOW

The Ronald O. Perelman Department of Dermatology and the Department of Cell Biology, NYU School of Medicine, New York*Address reprint requests to Seth J. Orlow, The Ronald O. Perelman Department of Dermatology, NYU School of Medicine, 560 FirstA�enue, Dermatology H-100, New York, NY 10016. E-mail: seth.orlow@med.nyu.edu

Received 27 February 2001; in final form 12 April 2001

limited to the eye, OA1 represents an ideal model systemO� cular a� lbinism type 1� (OA1) is an X-linked recessive disor-der characterized by a severe reduction of visual acuity, and to study the relationship between pigmentation and visual

development. Based upon sequence homology and bio-hypopigmentation of the retina that leads to nystagmus, stra-bismus, and photophobia/photodysphoria. Microscopic exami- chemical studies, OA1 may represent a novel intracellular

G-protein coupled receptor. Understanding the function ofnation of both retinal pigment epithelium and skinmelanocytes in OA1 reveals the presence of macrome- OA1 will contribute greatly to our understanding of

melanosome biogenesis and the role of pigmentation in visuallanosomes, suggesting that the OA1 gene product plays a rolein melanosome biogenesis. Studies of mutations identified development.from OA1 patients and an Oa1 knock-out mouse modelfurther implicate OA1 protein function in the late stage of Key words: Ocular albinism, Lysosome, Endosome, G-protein

coupled receptormelanosome development. Because its effects are primarily

tations (4, 6) necessitates an examination of family membersto confirm the diagnosis and for purposes of geneticcounseling.

Histologic Features of OA1

The histologic hallmark of OA1 is the presence of macrome-lanosomes or melanin macroglobules (MMGs, Fig. 1) inboth the eye and the skin of affected persons, even thoughtheir skin color appears to be normal. These organelles maybe seen by both light and electron microscopy in the skin ofmost affected males and many obligate heterozygote femalesand have also been noted upon pathologic examination ofthe retinal pigment epithelium (RPE) (7–9). Indeed, thepresence of MMGs on skin biopsy is often used to distin-guish OA1 from other forms of ocular albinism, althoughMMGs have been absent from skin biopsies from rarekindreds with apparent OA1 (10). In addition to theMMGs, a decreased number of normal melanosomes hasbeen noted (8, 11) in both the epidermis and the RPE.

INTRODUCTIONClinical Features

Ocular albinism type 1 (OA1), also referred to as Nettleship-Falls type of OA, is the most common form of ocularalbinism, with a prevalence in the United States estimated tobe 1/50,000 (1). The disease is inherited in an X-linkedrecessive fashion. Affected males exhibit all of the ocularstigmata of albinism, including reduction in visual acuity,nystagmus, strabismus, iris translucency, foveal hypoplasia,hypopigmentation of the retina, and loss of stereoscopicvision due to misrouting of the optic tracts (2). Carrierfemales, by contrast, have normal vision but examination oftheir retinal pigment epithelium reveals a patchy hypopig-mentation as a result of mosaic inactivation of the affectedX chromosome (3). The severity of OA1 is reported todepend upon the ethnic background of the affected person,being less severe in those of racial groups exhibiting verydark constitutive skin pigmentation than those more lightlypigmented (4, 5). The extreme variability in clinical manifes-

Abbre�iations – CHS, Chediak–Higashi syndrome; ER, endoplasmic reticulum; GPCR, G-protein coupled receptor; LE, late endosome;M6PR, mannose-6-phosphate receptor; MMG, melanin macroglobule; OA1, ocular albinism type 1; RPE, retinal pigment epithelium; TGN,trans-Golgi network; TM, transmembrane; TYR, tyrosinase; Tyrp1, tyrosinase-related protein 1

Pigment Cell Res. 14, 2001 243

MMGs are typically large spherical bodies, and lack thefilamentous substructure typical of normal melanosomes.Instead they contain numerous electron lucent vesicles em-bedded in a matrix of an amorphous electron dense materialpresumed to be melanin. The cortical area is often lesselectron dense, rich in vesicles and with a granular back-ground. MMGs with diameters up to 5 �m are not unusual(8, 11). The genesis of these structures is not yet known. Onetheory holds that they are a form of aberrant melanosomein which melanization takes place in a centrifugal pattern bytyrosinase (TYR) brought to the organelle by the manyvesicles that fuse with it. An alternative theory proposes thatthe MMGs are a form of autophagolysosome (12).

Gene Cloning and Protein Structure and Localization

Positional cloning led to the identification of the humanOA1 gene (13). Subsequently, Oa1, the murine homolog ofOA1, was cloned (14, 15) and showed extensive homologyto its human counterpart except in its carboxy terminus.OA1 was originally thought to be comprised of sixtransmembrane (TM) domains and to bear no homology toany previously reported protein (13, 14). Recently, however,OA1 has been suggested to be a member of the seven TMgroup of G-protein coupled receptors (GPCRs) by virtue ofits weak similarities to the members of families A, B and Eof the GPCR superfamily with the vasoactive intestinalpeptide receptor (VIPR) showing the closest homology (16).A GPCR-type model for OA1 predicts that the ligandbinding face of the protein has a lumenal orientation, andthat OA1 would couple with one or more G-proteins to linkto effector molecules such as adenylate cyclase in order totransduce a signal to the cytosol of the pigment cell. Thereis no precedent in any organism or cell type for a GPCR

with an exclusively intracellular location, transducing a lu-menal-to-cytosolic signal. However, since the homology be-tween OA1 and GPCRs is not very strong, identification ofthe ligand for OA1 would help to confirm its function as aGPCR. It is also possible that OA1 is not regulated by aligand, but is constitutively active or is even negativelyaffected by ligand binding.

The human OA1 and mouse Oa1 genes encode proteinswith predicted lengths of 404 and 405 amino acids, respec-tively. The human gene product of OA1 was originallyidentified as a 60 kDa glycoprotein derived from a 46–48kDa precursor (17). However, we have recently found thatfor murine Oa1, the 48 kDa form is the mature glycosylatedform which is altered to 44–46 kDa by treatment ofmelanocytes with tunicamycin or the protein with glycopep-tidase F (18). A melanosomal localization for OA1 wasreported based largely upon immuno-electron microscopy(16, 17). We examined the distribution of Oa1 by immu-nofluorescence and found that it is highly prevalent in theendolysosomal compartment of melanocytes (18). Upondensity gradient centrifugation of subcellular organelles ofmurine melan-a cells, the Oa1 protein colocalized with thelate endosomal/lysosomal protein LAMP-1, but minimaloverlap was observed with melanosomal proteins such asTYR and Tyrp1 in the high density region of the gradient.Furthermore, immunofluorescence staining revealed thatcolocalization of Oa1 with an authentic melanosomalprotein, Tyrp1, was limited to the perinuclear area repre-senting the Golgi/TGN (Fig. 2). In contrast, colocalizationof Oa1 with LAMP-1 was extensive. Like the keymelanogenic enzyme TYR, the expression of Oa1 protein bymelanocytes was stimulated by �-MSH and inhibited byAgouti signal protein (18). Thus, Oa1 is an integral mem-brane glycoprotein that is melanocyte specific and localizedin endolysosomes, but appears to be largely absent frommature melanosomes. In non-melanocytic cells such asCOS-1 and COS-7, both human OA1 and mouse Oa1displayed a vesicular distribution and colocalize with en-dolysosomal markers (16, 19).

Mutations Causing OA1 and the Cellular Distribution ofthe Mutant Proteins

OA1 gene mutations have been identified in many patientsfrom the US, Europe and Australia (4–6, 10, 20–23). MostOA1 alleles appear to be loss-of-function mutants. Largedeletions as well as missense substitutions that are locatedthroughout the central coding region (TM domains) havebeen found; a few are present at the N-terminus. Mutationshave not been identified within the less conserved C-termi-nal cytoplasmic domain. Notably, all disease-associated mis-sense mutations reported in patients with OA1 involveamino acid residues conserved in the mouse protein.

In our recent studies (19) we created a fusion proteinbetween Oa1 and green fluorescent protein (GFP) of thejellyfish Aequorea �ictoria. When expressed in melanocytes,essentially total overlap is seen between endogenous Oa1and Oa1-GFP (18) confirming that the tagged protein isappropriately recognized by the cellular trafficking machin-ery. Oa1-GFP thus represents a powerful tool for examining

Fig. 1. Electron micrograph of macromelanosome (MMG) fromskin biopsy of patient with OA1. Courtesy of Dr. Rhonda Schnur.

Pigment Cell Res. 14, 2001244

Fig. 2. Immunofluorescence localization of Oa1-GFP and Tyrp1 inmelan-a cells (methods as in Samaraweera et al.). Cultured melan-acells were fixed and labeled with MEL-5 antibody for Tyrp1 protein(red). Note that the colocalization of Oa1 with Tyrp1 is limited tothe perinuclear region. Bar=5 �m.

ing and subcellular distribution (see Table 1). Both studiesshowed that ER retention of the mutant proteins is a majormechanism for the pathogenesis of OA1. In the study byd’Addio et al. (see Table 1), the 19 OA1 mutants wereclassified into two biochemical groups based on their glyco-sylation pattern and correlation with their subcellular local-izations upon transfections in COS cells. Group I consists ofnormally-glycosylated mutants (Q124R, A138V, S152N,G229V, T232K, E235K, I244K, E271G) that showed agranular distribution, whereas group II (R5C, G35D,D78N, G84D, C116S, G118E, W133R, A173D, I261N,W292G, �T290) was comprised of the aberrantly glycosy-lated mutants that were retained in the ER, similar to theClass I mutant category defined in our study. A correlationbetween a lack of glycosylation and ER retention was lessclear when mouse Oa1 is transfected into COS cells, and wehave found that the glycosylation patterns of several ClassII mutants are indistinguishable from the pattern exhibitedby wild-type Oa1 protein (Shen and Orlow, unpublisheddata).

Clues to the Function of the Oa1 Protein

The use of a panel of endolysosomal markers revealedpossible functions of the Oa1 protein (19). Expression ofWT Oa1-GFP and Class III mutants in COS cells inducedenlargement of the diameter of perinuclear LAMP-2-posi-tive LEs when compared with cells transfected with theER-retained Class I mutants. Measurements of theseLAMP-2 positive LEs revealed 23% increase in the averagediameter in cells transfected with WT Oa1-GFP (1.72�0.83�m) vs. G35D, which was retained in the ER (1.4�0.44�m). This difference in diameter was statistically significantat P�0.0001, and corresponded to an 86% increase in thevolume per organelle. In addition, there was an 88% in-crease in the number of these LEs in COS cells transfectedwith WT Oa1, which contained an average of 15, as com-

Table 1. Classifications of the missense mutations identified inpatients with ocular albinism

Subcellular distribution

OA1 mutant Group (24)Class (19)

R5C — ER (II)G35D ER (I) ER (II)

—ER (I)L39RER (II)D78V (N) ER/lysosomal (II)ER (II)ER/lysosomal (II)G84D (R)

ER/lysosomal (II) ER (II)C116R (S)G118E ER/lysosomal (II) ER (II)Q124R — Lysosomal (I)W133R endo/lysosomal (III) ER (II)

Lysosomal (I)endo/lysosomal (III)A138Vendo/lysosomal (III) Lysosomal (I)S152N

A173D ER/lysosomal (II) ER (II)G229V — Lysosomal (I)

Lysosomal (I)T232K endo/lysosomal (III)E235K endo/lysosomal (III) Lysosomal (I)I244K — Lysosomal (I)I261N — ER (II)E271G — Lysosomal (I)

—�T290 ER (II)ER (II)W292G ER/lysosomal (II)

the cell biology of Oa1. Based upon the mutations reportedto cause OA1 (20–23), we constructed 13 plasmids encodingmurine Oa1-GFP where each of those DNA sequences alsoencodes a single amino acid substitution that has beenreported in an actual patient. Subcellular localization of thewild type and mutated versions of Oa1-GFP was comparedwith different organelle markers. Upon transfection intoCOS cells, wild type (WT) Oa1-GFP is localized to lateendosomal/lysosomal compartments. Mutations could begrouped into three classes according to their intracellulardistribution (see Table 1): Class I, endoplasmic reticulum(ER) retention mutants, is composed of missense mutationsat the N-terminus of the Oa1 protein, G35D and L39R;Class II mutants, including D78V, G84R, C116R, G118E,A173D, and W292G, often are present in small peripheralLAMP-2-positive granules in addition to ER; Class IIImutants: W133R, A138V, S152N, T232K, and E235K, sim-ilar to WT Oa1-GFP, localize to LE/lysosomal compart-ments. Similar results were obtained by d’Addio et al. (24),by expressing wild-type human OA1 and 19 missense muta-tion constructs in COS-7 cells and examining their process-

Pigment Cell Res. 14, 2001 245

Table 2. Enlargement of LEs in COS cells by expression of WTOa1-GFP and Class III mutants but not by Class I or Class IImutantsa

Oa1-GFP Relative volume ofAverage diameter inconstructs �m (number of LE compartment/cell

organelles analyzed)

Experiment AWT Oa1-GFP 27.61.72 (n=1010)Class I 8.01.4 (n=494)mutant (G35D)

Experiment BClass I 1.44 (n=712) 14.2mutant (L39R)Class II 15.21.35 (n=932)mutant (C116R)Class III 1.67 (n=1360) 42.4mutant (S152N)

a COS cells transfected with different Oa1-GFP constructs wereanalyzed by immunofluorescence microscopy. Two separate experi-ments were performed. Experiment A compared the effect of WTand Class I mutant G35D Oa1-GFP on enlargement of LEs. TheP-value of WT compared with G35D is �0.0001. Experiment Bcompared the enlargement effect among three Classes of mutants.The P-values of Class II mutant C116R and Class III mutantS152N compared with Class I mutant L39R are each�0.0001. Thediameter of individual LAMP-2-positive vesicles was measured andexpressed in �m. Number in parentheses indicates the number oforganelles analyzed for the measurements. (Adapted in part fromShen, Rosenberg, Orlow. Traffic 2001, 202–211.)

COS cells are dispersed throughout the cell without signifi-cant clustering. This dramatic difference in distribution mayexplain the reduced number of granules positive for M6PRsince the redistribution of the larger granules in the perinu-clear region maybe the result the clustering of the smallM6PR positive granules.

Four of the Class III mutants (W133R, A138V, S152N,and T232K) represent mutations in patients known to haveMMGs upon skin biopsy (23). This suggests that althoughClass III mutants traffic correctly to the endolysosomalcompartment and retain the ability to cause enlargement ofLE diameter, the loss of an additional function of OA1 islikely to be responsible for the presence of MMGs (andalbinism) in persons bearing this class of mutations, andcorrelates with the lack of effect of these mutants on M6PRdistribution.

Two mutants (D78N and C116S) were tested by Schi-affino and coworkers for their ability to bind GPCR associ-ated subunits G� and G�i (16). In this study,co-immunoprecipitation revealed that the mutant proteininteracted either less efficiently or did not bind to G proteinsubunits (respectively) in contrast to the wild-type OA1protein. Since the missense mutations D78N and C116S inOA1 affect highly conserved residues critical for the func-tion of most GPCRs (25), this seems to provide support thatOA1 indeed functions as an intracellular GPCR. However,the observation (19, 24) that a single amino acid change canalter the conformation of the protein and prevent it fromexiting the ER weakens this argument. The fact that ER-re-tained mutants (including Q124R, A138V, S152N, G229V,T232K, E235K, I244K, E271G) did not reveal any differ-ence in G protein-binding ability when compared with wild-type OA1 (24) suggests that the ability to bind G proteinsubunits is not frequently disrupted by albinism-causingmutations unlike the intracellular routing of OA1.

Comparable to the MMGs described in OA1 patients,histological analysis of the RPE from mice with targeteddeletion of the Oa1 gene also reveal the present of MMGs(26). During embryonic development, MMGs are not de-tectable in the RPE of mutant mice, but as the newbornmice mature to 7 days post-partum, a majority of themelanosomes displayed a giant phenotype, even though thenumber of melanosomes per cell is reportedly constant (26).Ultrastructural analysis of the RPE in mutant Oa1 miceallowed the identification of a central core region suggestiveof the structure of a normal melanosome. The lack ofevidence of intermediate structures showing melanosome-melanosome fusion suggested that MMGs may be the resultof abnormal growth of single melanosomes (26). However,whereas normal melanocytes in human skin contain manymelanosomes, only one or a few MMGs are typically foundper melanocyte in skin biopsies from persons with OA1,accompanied by a decreased number of normalmelanosomes (8, 11). This suggests that each MMG in OA1is formed at the expense of several melanosomes. Further-more, if mature pigmented melanosomes become MMGs, itis then difficult to explain the fundus hypopigmentation ofOA1 since total melanin should not be reduced according tothis hypothesis (26). Regardless of their origin, it is not clearwhether the diminution in the number of normal

pared to cells expressing G35D, which contained an averageof eight such organelles (P�0.0001). Importantly, whenthese two observations were combined, it was determinedthat the total volume of the multivesicular LE compartmentin WT transfected cells was 3.5-fold larger than the volumein cells transfected with G35D (summarized in Table 2).These results suggest that WT Oa1 has the capacity to alterthe LE compartment. In a similar experiment, the relativevolumes of the LE compartment in COS cells expressingC116R (a Class II mutant located in the ER and lysosomesbut not LE) or S152N (a Class III mutant with a distribu-tion similar to WT) were compared to the volume of the LEcompartment in cells expressing L39R (another Class IER-retained mutant). When S152N was compared to L39Rin this experiment, it resulted in both a 16% increase indiameter per LE (1.67�0.73 �m) and a 91% increase in thenumber of LEs per cell (27.2). This indicated that the totalvolume of the LE compartment in cells expressing S152Nwas 3-fold larger than the volume of controls expressingL39R. Thus, a Class III mutant protein retained the capac-ity demonstrated by WT Oa1 to significantly enlarge the LEcompartment (summarized in Table 2).

Interestingly, we observed that COS cells transfected withWT Oa1-GFP exhibited diminished numbers and altereddistribution of granules immunoreactive for mannose-6-phosphate receptor (M6PR) when compared to control cellsor to cells expressing Oa1 mutants (19). None of the 13mutants examined displayed the full range of effects on theredistribution of mannose-6-phosphate receptor exhibited byWT Oa1. In cells transfected with WT Oa1-GFP, there is areduced number of larger M6PR positive granules clusteredin the perinuclear region. Small M6PR positive granules incells transfected with mutant Oa1-GFP or untransfected

Pigment Cell Res. 14, 2001246

melanosomes replaced by these giant organelles is responsi-ble for the albino phenotype (11).

Models for OA1 Function

OA1 may control a step in melanosomal sorting ortrafficking from late endosomesNascent polypeptides are inserted into the ER where theybegin to undergo co- and post-translational modification.Properly folded proteins that pass the ER quality-controlmechanism enter the Golgi where they undergo furthermodification. They then pass on to the trans-Golgi network(TGN), where they are sorted to endolysosomal pathwaysbased upon the signals in their cytoplasmic domains (27,28). Molecular sorting in the late endosomal compartmentmay target proteins to specialized organelles such as lyso-somes and melanosomes (Fig. 3). We hypothesize thatmelanosomal proteins such as tyrosinase and/or Tyrp1 maystart their journey with OA1 from the TGN to melanosomesvia an endosomal compartment (Fig. 3). Melanosomal andlysosomal proteins share the same pathway at this earlystage (29–31), and OA1 may serve to mark or control abranch point to the melanosome. Unlike tyrosinase, how-ever, OA1 may be a resident protein of the late endosomal/lysosomal compartment, controlling budding from this‘premelanosomal’ compartment. In its absence, the late en-dosomal compartment continues to enlarge to form themultivesicular appearance characteristic of the MMGs.

This model could explain the fact that in OA1 patients,the eyes are more severely affected than the skin. RPE cellsspecialize in degrading the shedded tips of photoreceptorrod outer segments, and it has been shown thatmelanosomes are connected to lysosomal pathways in RPEcells (32). Thus, OA1 may function in controlling traffickingout of the endolysosomal compartment to melanosomes,and this function may be more critical in RPE than incutaneous or follicular melanocytes since the lysosomalpathway is so highly active in RPE.

Giant organelles similar to those found in OA1 patientshave also been observed in Chediak–Higashi syndrome(CHS) (33) and in beige mice. Lyst, the gene productdefective in these conditions, is also thought to be involvedin intracellular membrane trafficking to a number of lyso-some-related organelles including melanosomes (33, 34). Thefact that only a specific subset of cell types and tissues areaffected in patients with such multi-organelle disorders sug-gests that there are multiple alternative pathways of organel-lar cargo-protein trafficking, and perhaps a certain degree ofredundancy or leakiness permitting sufficiently normal or-ganellar biogenesis in some but not all tissues. Our modelfor OA1 function may reflect one of the many possiblepathways.

Recently, based upon electron microscopic studies on theretinal pigment epithelium of Oa1 knock-out mice (26), ithas been suggested that Oa1 plays a role in the final stagesof melanosome growth and maturation. This would beconsistent with our hypothesis that OA1 may play a role inregulating the LE/lysosomal compartment that serves as anintermediate step for protein sorting to the melanosome.However, the observation of normal melanosomes appear-ing first, followed by MMGs later, suggests that the MMGsmight indeed arise from continued vesicular fusion withmelanosomes. In other words, OA1 might normally functionto inhibit or otherwise regulate vesicular budding from theLE or their fusion with melanosomes.

OA1 as a ‘sensor’ of melanin, melanin intermediates, ormelanosomal maturityMutations in multiple genes that decrease eye pigmentationall share the same developmental eye defects demonstratingthat a melanin-related agent is crucial for normal develop-ment of the visual system (35). This function has seriousimplications for neurogenesis during eye development andmight explain the misrouting of optic fibers and retinaldistribution linked with ocular albinism. OA1 has beenproposed as a perfect candidate for this sensor systembecause the hypopigmentation in OA1 is largely limited toocular tissues, especially the retinal pigment epithelium(RPE), even though microscopic abnormalities inmelanosomes are observed in both eye and the skin. Fur-thermore, the possible function of OA1 as a GPCR in-creases the likelihood of OA1 as a sensor and consequentlya signal transducer. Possible ligands for OA1 include smallmolecules such as tyrosine, Dopa, and other melanin inter-mediates or even large molecules such as melanin polymer(36), or tyrosinase. Thus, in OA1, even though the ability tomake melanin is preserved, the absence of a sensor functionmight result in a delay in retinal maturation. Alternatively,

Fig. 3. A model of OA1 function in endocytic traffic and biosyn-thetic delivery to melanosomes. Newly synthesized melanosomalproteins exit the TGN. The melanosomal delivery pathway maypartially overlap with the clathrin-mediated route for delivery ofnewly synthesized, mannose-6-phosphate-tagged lysosomal enzymessuch as acid hydrolases. Arrows indicate the recycling of mannose-6-phosphate-receptor (M6PR) between TGN and endosomal com-partments. The late endosomal (LE) compartment is the proposedsorting point for lysosomes and melanosomes, where OA1 may playa role. Mutations in OA1 block (��) the branch point tomelanosomes from the late endosome. Abbreviations: EE, earlyendosome; RE, recycling endosome; MVB, multivesicular body;M6PR, mannose-6-phosphate receptor; TYR, tyrosinase; LE, lateendosome.

Pigment Cell Res. 14, 2001 247

a feedback mechanism might exist where in the presence ofa mutant OA1, accumulation of ‘unsensed’ melanin leads toa series of events that could inhibit RPE cell growth (37,38). Discovery of the potential ligand and downstreameffectors of OA1 will be necessary steps in understanding itsfunction.

Acknowledgements – The authors thank Drs. Dorothy Bennett andRivka Rachel for helpful discussions. BS was supported in part byPHS training grant 5T32AR07190 to the Department of Dermatol-ogy, NYU. This work was supported in part by PHS grantsEY10223 and AR41880 to SJO.

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