Báo cáo khoa học: N-Glycosylation is important for the correct intracellular localization of HFE and its ability to decrease cell surface transferrin binding pptx

combinations of double mutants, which displayed alower apparent molecular mass than either wild-type or single mutant forms of the protein.. To test this, we transfected HEK293T cells to

localization of HFE and its ability to decrease cell surface transferrin binding

Lavinia Bhatt1, Claire Murphy2, Liam S.O’Driscoll2, Maria Carmo-Fonseca3, Mary W McCaffrey1 and John V Fleming2,3

1 Department of Biochemistry, Biosciences Institute, University College Cork, Ireland

2 Department of Biochemistry, School of Pharmacy and ABCRF, University College Cork, Ireland

3 Institute of Molecular Medicine, University of Lisbon, Portugal

Keywords

HFE; N-glycosylation; transferrin; transferrin

receptor 1; b2-microglobulin

Correspondence

J V Fleming, Department of Biochemistry

and School of Pharmacy, University College

Cork, Cork, Ireland

Fax: +353 21 4901656

Tel: +353 21 4901679

E-mail: j.fleming@ucc.ie

Note

L Bhatt and C Murphy contributed equally

to this work

(Received 8 February 2010, revised 14 May

2010, accepted 2 June 2010)

doi:10.1111/j.1742-4658.2010.07727.x

HFE is a type 1 transmembrane protein that becomes N-glycosylated dur-ing transport to the cell membrane It influences cellular iron concentra-tions through multiple mechanisms, including regulation of transferrin binding to transferrin receptors The importance of glycosylation in HFE localization and function has not yet been studied Here we employed bio-informatics to identify putative N-glycosylation sites at residues N110, N130 and N234 of the human HFE protein, and used site-directed muta-genesis to create combinations of single, double or triple mutants Com-pared with the wild-type protein, which co-localizes with the type 1 transferrin receptor in the endosomal recycling compartment and on dis-tributed punctae, the triple mutant co-localized with BiP in the endoplas-mic reticulum This was similar to the localization pattern described previously for the misfolding HFE-C282Y mutant that causes type 1 hered-itary haemachromatosis We also observed that the triple mutant was func-tionally deficient in b2-microglobulin interactions and incapable of regulating transferrin binding, once again, reminiscent of the HFE-C282Y variant Single and double mutants that undergo limited glycosylation appeared to have a mixed phenotype, with characteristics primarily of the wild-type, but also some from the glycosylation-deficient protein There-fore, although they displayed an endosomal recycling compartment/punc-tate localization like the wild-type protein, many cells simultaneously displayed additional reticular localization Furthermore, although the majority of cells expressing these single and double mutants showed decreased surface binding of transferrin, a number appeared to have lost this ability We conclude that glycosylation is important for the normal intracellular trafficking and functional activity of HFE

Structured digital abstract

l MINT-7896236 , MINT-7896218 : beta2M (uniprotkb: P61769 ) physically interacts ( MI:0915 ) with HFE (uniprotkb: Q30201 ) by anti bait coimmunoprecipitation ( MI:0006 )

l MINT-7896162 : TfR1 (uniprotkb: P02786 ) and HFE (uniprotkb: Q30201 ) colocalize ( MI:0403 )

by fluorescence microscopy ( MI:0416 )

Abbreviations

ER, endoplasmic reticulum; ERC, endosomal recycling compartment; HH, hereditary haemachromatosis; b2M, b2 microglobulin; MHC, major histocompatability complex; PNGase F, N-glycosidase F; Tfn, transferrin; TfR1, transferrin receptor 1; TfR2, transferrin receptor 2.

The hereditary haemochromatosis (HH) protein HFE

(high Fe) is a type 1 transmembrane protein that plays

an important role in controlling physiological iron

homeostasis [1–3] It is widely expressed throughout

the body with expression highest in cells that are

involved in iron metabolism [4–6] Mutations in the

HFE protein cause type 1 HH, which is an inherited

disease of iron metabolism that results in iron overload

in several organs [4,7] The HFE mutation detected in

the majority of HH patients results in the replacement

of cysteine residue 282 with tyrosine (C282Y) The

mutant protein is unable to form a structurally

impor-tant disulfide bridge required for HFE interactions

with b2 microglobulin (b2M) [4,5,8–11] In the absence

of b2M binding, the protein misfolds and is retained in

the endosplasmic reticulum (ER) where it induces an

unfolded protein stress response that is characterized

by alternative splicing of XBP-1 and increased

expres-sion of CHOP and BiP [12–14] A second

well-described HFE mutation associated with HH leads to

the replacement of histidine at residue 63 with

aspar-tate This mutant is capable of b2M interaction and

cell-surface expression but is unable to regulate cellular

iron uptake like the wild-type HFE protein [4,15]

Although much insight into HFE function has been

gained through studying the cellular and biochemical

properties of these different mutant proteins, the exact

mechanism by which HFE regulates intracellular iron

levels is still not completely understood

The HFE primary sequence exhibits significant

homology to major histocompatability complex

(MHC) class I molecules and the protein is organized

into a1, a2 and a3 structural domains that resemble

those described for MHC class I and related proteins

[4,16] The N-terminal a1 and a2 domains come

together to form a superstructure composed of two

a helices layered on top of eight anti-parallel b sheets

In MHC class I proteins this a1/a2 superstructure

forms a peptide-binding groove that mediates antigen

binding and presentation to CD8+ cytolytic T cells

In HFE, the proximity of the two a helices and the

presence of amino acid side chains that project into

the groove appear to prevent peptide binding [16] The

a3 region, like its homologous domain in MHC

class I, is an immunoglobulin-like domain that medi-ates binding to b2M [16,17] C-Terminal residues of HFE mediate its retention in the cell membrane Shortly after HFE was discovered it was reported to co-localize and interact with the type 1 transferrin receptor (TfR1) [5,18] TfR1 mediates the endocytosis

of iron-loaded transferrin into acidic endosomes where the iron is released and transported into the cytoplasm via the Nramp2-DCT1 iron transporter Apo-transfer-rin and TfR1 are recycled to the cell surface where apo-transferrin is released [3,19] Crystallography stud-ies suggest that the a3 stem of HFE lstud-ies parallel to the cell membrane and that the a1/a2 superstructure inter-acts with helical regions located within TfR1 In this way, it is possible for two HFE proteins to be posi-tioned at either side of the TfR1 homodimer and form

a tetrameric complex that exhibits twofold symmetry [17] Reports from crystallography experiments have been supported by mutagenesis studies that identified residues located at the end of an a-helical region of the HFE a1 domain (V100 and W103A) as being of par-ticular importance for TfR1 interactions [16,17,20] The effect of HFE binding to TfR1 is to lower the affinity of the receptor for transferrin [15] This most likely reflects the existence of overlapping HFE and transferrin-binding sites on the receptor [21,22] Suc-cessive studies indicate that HFE and TfR1 co-localize during endosomal trafficking, although there are con-tradictory reports as to whether TfR1 recycling is affected by HFE [23–29]

Despite these well-described interactions, there is mounting evidence that HFE regulation of cellular iron levels may not depend solely on TfR1 binding [30,31] Attention has shifted to a second transferrin receptor, TfR2, whose pattern of expression is more restricted than that of ubiquitously expressed TfR1 [32] Levels of TfR2 are highest in hepatocytes, the predominant site of HFE expression, and recent stud-ies have confirmed that the two proteins are capable of interacting [33,34] The nature of these interactions dif-fers from those observed between HFE and TfR1 in that they are mediated by the a3 domain of HFE, as opposed to the a1/a2 superstructure [33] An emerging model, therefore, is that TfR2 competes with TfR1 for

l MINT-7896258 , MINT-7896317 , MINT-7896330 , MINT-7896348 , MINT-7896366 : HFE (uni protkb: Q30201 ) and transferrin (uniprotkb: P02787 ) colocalize ( MI:0403 ) by fluorescence microscopy ( MI:0416 )

l MINT-7896149 : HFE (uniprotkb: Q30201 ) and BiP (uniprotkb: P11021 ) colocalize ( MI:0403 )

by fluorescence microscopy ( MI:0416 )

HFE binding This occurs maximally at high

concen-trations of transferrin The resulting HFE–TfR2

com-plex, which is stabilized at high iron concentrations, is

believed to somehow regulate the expression of other

genes involved in iron metabolism This includes

hepci-din, a 25 amino acid antimicrobial peptide that is

expressed in liver cells and is now recognized as a key

regulator of iron homeostasis in the body

Hepatocel-lular hepcidin mRNA levels have been shown to be

regulated by HFE, and are altered in

haemachromato-sis patients with the C282Y mutation [35–37]

The importance of N-glycosylation with respect to

protein expression and function is highly variable

Roles have been described in the secretion, stability

and oligomerization of proteins [38,39], the

bioactivi-ties of enzymes [40] and the binding affinibioactivi-ties of

ligands and receptors [41] In many instances, specific

functions can be attributed to glycosylation at specific

sites For example, the human gonadotropin a subunit

has N-glycosylation sites at residues Asn52 and Asn78

that have been shown to differentially regulate receptor

signalling and secretion, respectively [38,39] Another

example is the type 1 transferrin receptor, which has

N-glycosylation sites at residues Asn251, Asn317 and

Asn727 Mutation of Asn727 decreases cell-surface

expression, whereas mutation at the other two sites

does not [42]

HFE becomes glycosylated during post-translational

processing Transfection studies have confirmed that

this involves N-glycosylation, and incubation of lysates

from HFE-expressing cells with N-glycosidase F

(PNGase F) leads to the accumulation of lower

molec-ular mass HFE proteins [13,43] The carbohydrate

moiety undergoes processing and

endoglycosi-dase H-resistant HFE isoforms can be detected by

30 min post translation [10,13,18,23] Although these

studies demonstrate that HFE is glycosylated, the

specific role, if any, that glycosylation might play in

cellular HFE function has not previously been studied

In this article, we map the sites of HFE

N-glycosyla-tion and examine the importance of glycosylaN-glycosyla-tion on

parameters of protein localization and function

Results

Tunicamycin treatment results in a reticular

pattern of HFE localization

Previous studies have demonstrated that HFE

under-goes post-translational N-glycosylation As a first step

towards assessing the importance of N-glycosylation

on HFE expression, we transiently transfected HuTu80

to express HFE-WT–HA and cultured the cells in the

presence or absence of tunicamycin to inhibit glycan production Control, untreated cells predominantly exhibited a punctate pattern of HFE expression with a tubulovesicular concentration in the pericentrosomal region (Fig 1A,D), consistent with previous observa-tions [11,28] Immunostaining with TfR1, anti-Rab11a and Rab11-FIP3 Ig has identified the HFE-containing pericentrosomal compartment of HuTu80 cells as the endosomal recycling compartment (ERC) [28] Treatment of HFE-WT–HA-expressing cells with tunicamycin altered this pattern of localization and resulted in a reticular pattern of cell localization (Fig 1B,D) Immunostaining showed significant co-localization with the ER chaperone protein BiP, demonstrating that the HFE-WT–HA was now locali-zing primarily to the ER (Fig 1B,D)

A similar pattern of reticular expression and BiP co-localization was observed when HuTu80 cells were transfected to express the HH-causing HFE-C282Y variant (Fig 1C,D), which has been shown through multiple biochemical and microscopy approaches to be retained in the ER [10,13,18,28,29]

HFE is glycosylated at residues Asn110, Asn130 and Asn234

Although the results in Fig 1 point towards an impor-tant role for glycosylation in HFE localization, it remains possible that the effects of tunicamycin treat-ment were indirect To directly examine the impor-tance of glycosylation on HFE, it was necessary to generate an N-glycosylation-deficient mutant To this end, we used a bioinformatic prediction program (netnglyc 1.0 Server; Technical University of Den-mark) to identify putative glycosylation sites in the protein Consistent with previous predictions [18], we identified three high-probability sites: asparagines at positions 110, 130 and 234 Starting with wild-type HFE, we generated all possible combinations of single and double putative N-glycosylation site mutants using site-directed mutagenesis The wild-type and mutant expression constructs were transfected into HEK293T cells and the lysates analysed by western blotting

The results from these experiments, which are shown

inFig 2A, indicate that the introduction of single ala-nine mutations at N110, N130 and N234, respectively, resulted in the production of HFE proteins that migrated with increased mobility on SDS/PAGE com-pared with the wild-type This suggested that all three sites in the wild-type proteins are capable of becoming glycosylated The decrease in apparent molecular mass became even more pronounced for proteins containing

combinations of double mutants, which displayed a

lower apparent molecular mass than either wild-type

or single mutant forms of the protein

Although immunoblot analysis demonstrated that

the single and double mutants had decreased mass

compared with the wild-type protein, they still

appeared to be of higher molecular mass than the

unglycosylated form of the wild-type HFE protein –

which was produced when wild-type-expressing cells

were treated with tunicamycin (Fig 2A; WT-Tunica)

This suggested that both the single and double

mutants were still partially glycosylated To test this,

we transfected HEK293T cells to express either the

wild-type or mutant proteins, and incubated the cells

in the presence or absence of tunicamycin Drug treatments resulted in the accumulation of forms of the mutant proteins that were of lower apparent molecular mass and of similar size to the unglycosy-lated form of the wild-type protein (see Fig 2B for single mutants and Fig 2C for double mutants) This suggested that the mutant proteins do indeed still undergo limited glycosylation For the single mutants, additional supporting evidence for the per-sistence of N-linked glycans was obtained by PNG-ase F digestions of immunoprecipitated proteins, which then migrated with lower apparent molecular mass compared with the undigested forms (results not shown)

A

B

C

D

Fig 1 Inhibition of N-glycosylation influ-ences patterns of HFE intracellular localiza-tion HuTu80 cells were transfected with constructs expressing the HFE-WT–HA (A, B) and HFE-C282Y–HA (C) proteins, HFE-WT-expressing cells were incubated for

1 h in the absence (A) or presence (1B) of

2 lgÆmL–1tunicamycin (Tunica) as indicated Cells were immunostained with anti-HA and anti-BiP Ig, and processed for fluorescence microscopy Co-localization masks were created as described in Materials and meth-ods, and represent areas with overlapping green and red pixels converted to white Scale bar, 10 lm Identical results were obtained when cells were transfected with constructs directed to express amino-tagged GFP–HFE-WT and GFP–HFE-C282Y, and in general we found that HA and GFP tags could be interchanged without altering the pattern of cell localization (data not shown) Figures shown are representative of at least three independent experiments (D) Graph showing the relative amounts of transfected cells exhibiting punctate or reticular localiza-tion of expressed HFE proteins (n = 3).

HFE NNN110/130/234/AAA triple mutant is

glycosylation deficient

To investigate whether the three N-glycosylation sites

studied to date are the only sites of HFE

N-glycosyla-tion – and with the aim of producing an HFE mutant that is completely deficient in N-glycosylation – we used site-directed mutagenesis to create a NNN110/ 130/234AAA triple mutant To determine the effect of these combined mutations on HFE, we transfected HEK293T cells to express wild-type, single, double or triple mutants

Western blot analysis of cell lysates shown inFig 3A indicated that the triple mutant fractionated with a lower molecular mass than the wild-type protein, and either the single or double mutants This lower molecu-lar mass form appeared to be the same size as the unglycosylated form of the wild-type protein produced

in tunicamycin-treated cultures (Fig 3A; WT-Tunica) These data suggested that all potential glycosylation sites had been mutated To confirm this, we transfected HEK293T cells to express wild-type or triple mutant forms of HFE and incubated the cells in the presence and absence of tunicamycin Drug treatment resulted

in the accumulation of an unglycosylated lower molec-ular mass form of the wild-type HFE protein, whereas

it had no effect on the apparent molecular mass of the triple mutant (Fig 3B)

In a second approach, HFE was immunoprecipitated from wild-type or triple-mutant-expressing cells and incubated with PNGase-F As is shown in Fig 3C, enzyme treatment of wild-type HFE resulted in the production of a lower molecular mass product Treat-ment had no detectable effect on migration of the tri-ple mutant, which had the same apparent molecular mass as the PNGase F-treated wild-type protein We conclude that HFE is normally glycosylated in vivo at three sites (N110, N130 and N234), and that mutation

of these sites gives rise to an HFE protein that is N-glycosylation deficient

N-Glycosylation of HFE is required for its appropriate localization to the ERC Our results from Fig 1 indicated that tunicamycin treatment of HFE-WT-expressing cells results in a reticular localization pattern To definitively establish the importance of N-glycosylation on HFE localization

in HuTu80 cells, we transfected cells to express GFP-tagged forms of wild-type or triple-mutant HFE As shown in Fig 4A, HFE-WT–GFP localized predomi-nantly to a tubulovesicular structure near the nucleus with some punctate staining, similar to that observed

in Fig 1A The GFP-tagged triple mutant, by contrast, predominantly displayed a reticular localization pat-tern (Fig 4A,B) This was similar to the expression pattern previously observed for HFE-WT in tunica-mycin-treated cells (Fig 1B)

A

B

C

Fig 2 Characterization of N-glycosylation site single and double

mutants (A) HEK293T cells were transfected to transiently express

WT–HA, N110–HA, N130A–HA, N234A–HA,

HFE-NN110/130AA–HA, HFE-NN130/234AA–HA or HFE-NN234/110AA–

HA proteins HFE-WT–HA expressing cells were incubated for 16 h

before lysis in the presence or absence of 1 m M tunicamycin (Tunica).

Cleared cell lysates were fractioned by 11% SDS/PAGE for

immuno-blotting with a mouse anti-HA Ig (B) Transiently transfected HEK293T

cells expressing HFE-WT–HA, HFE-N110–HA, HFE-N130A–HA or

HFE-N234A–HA were incubated for 16 h in the presence or absence

of 1 m M tunicamycin as indicated (Tunica) HA-tagged proteins in

cleared cells lysates were detected by immunoblotting with a mouse

anti-HA Ig (C) Transiently transfected HEK293T cells expressing

WT–HA, NN110/130AA–HA, NN130/234AA–HA or

HFE-NN234/110AA–HA protein were incubated for 16 h in the presence or

absence of 1 m M tunicamycin as indicated (Tunica) HA-tagged

pro-teins in cleared cells lysates were detected by immunoblotting with a

mouse anti-HA Ig.

HuTu80 cells transfected with HFE-WT or HFE

tri-ple-mutant proteins were subsequently immunostained

with an anti-TfR1 Ig The results from these

experiments demonstrated that the wild-type protein

co-localizes with TfR1 predominantly in the

tubulove-sicular perinuclear ERC and discrete punctae

(hereafter referred to as ERC/punctate pattern of

localization) The triple mutant, by contrast, shows no TfR1 co-localization (Fig 4A)

Although these results attest to the importance of N-glycosylation for the normal cellular localization of HFE, we wondered whether this was a cumulative effect or whether there are specific glycosylation sites that are more important than others for ensuring expression and recycling of the protein To test this,

we transfected HuTu80 cells to express HFE-N110A– GFP, HFE-N130A–GFP or HFE-N234A–GFP pro-teins In all instances, we observed that the majority of transfected cells displayed an ERC/punctate pattern of localization, similar to the wild-type protein (Fig 4A,B) Interestingly, a significant number of cells displaying this ERC/punctate pattern simultaneously displayed reticular localization in the same cells (50 ± 2% of N110A-expressing cells, 50 ± 2% of N130A-expressing cells and 58 ± 5% of N234A-expressing cells, n = 3) This was a feature also of cells expressing HFE double mutants, in which we like-wise observed an ERC/punctate localization pattern in the majority of cells (Fig 5A,B) and a significant num-ber of these cells simultaneously displaying reticular localization (55 ± 7% of NN110/130AA-expressing cells, 51 ± 4% of NN130/234AA-expressing cells and

67 ± 7% of NN234/110AA-expressing cells, n = 4) This type of mixed phenotype, where reticular locali-zation was observed in cells that already had the cor-rect ERC/punctate pattern, was not observed to any significant degree in cells expressing the wild-type or triple-mutant proteins

N-Glycosylation is important for interactions with b2M

The wild-type HFE protein interacts with b2M during transport to the cell surface It is commonly reported that misfolding and ER retention of the HFE-C282Y variant happens specifically because this b2M interac-tion does not occur [5,9–11] The HFE triple mutant, just like the HFE-C282Y mutant, shows a reticular pattern of localization and in immunostaining studies was seen to co-localize in the ER with BiP (Fig 6A) Accordingly, we wondered whether this pattern of localization reflected an underlying inability to interact with b2M or whether, in addition to b2M binding, the HFE protein needs to be appropriately glycosylated in order to successfully transit the ER HEK293T cells were transfected to express wild-type or mutant forms

of the HFE protein Cells were lysed and an anti-b2M

Ig used to immunoprecipitate b2M and any interacting proteins As shown in Fig 6B, the triple mutant showed significantly decreased interactions with b2M

A

B

C

Fig 3 Characterization of N-glycosylation site triple mutant (A)

HEK293T cells were transiently transfected with constructs

expressing HFE-WT–HA, HFE-N110–HA, HFE-N130A–HA,

HFE-N234A–HA, HFE-NN110/130AA–HA, HFE-NN130/234AA–HA,

HFE-NN234/110AA–HA or HFE-NNN110/130/234–HA (Triple)

pro-teins HFE-WT–HA expressing cells were incubated for 16 h before

lysis in the presence or absence of 1 m M tunicamycin (Tunica).

HA-tagged proteins were detected by fractionation of cleared cell

lysates on 11% SDS/PAGE for immunoblotting with a mouse

anti-HA Ig (B) Transiently transfected HEK293T cells expressing

HFE-WT–HA or HFE-NNN110/130/234–HA (Triple) proteins were

incubated for 16 h in the presence or absence of 1 m M tunicamycin

(Tunica) as indicated (C) HFE-WT–HA or HFE-NNN110/130/

234AAA–HA proteins were transiently expressed in HEK293T cells.

Forty-eight hours after transfection cells were harvested and

HA-tagged proteins were immunprecipitated with a polyclonal rabbit

anti-HA Ig Immunoprecipitated proteins were digested with

PNG-ase F and fractionated by 11% SDS/PAGE for immunoblotting with

a mouse anti-HA Ig.

This deficiency could not be attributed to a specific

glycosylation site, because each of the single mutants

retained the ability to interact with b2M The

HFE-C282Y mutant was employed in these experiments as a

negative control (Fig 6B)

Combined with our earlier cell localization results,

the data presented in Fig 6 point towards an

important role for N-glycosylation in HFE folding

However, once again, it is only when all three sites

are mutated that we observe a significant loss of

function

N-Glycosylation is important for HFE regulation

of transferrin binding Previous reports have established that in certain cell types HFE acts to regulate intracellular iron levels by decreasing the binding of transferrin to transferrin receptors and reducing cellular iron uptake as a conse-quence Indeed, it is commonly believed that iron over-load in HH occurs because the misfolding HFE-C282Y variant fails to make it to the cell surface and

is unable to exert this control We wanted to determine

Fig 4 Intracellular localization of HFE triple and single N-glycosylation site mutants (A) HuTu80 cells were transfected to express HFE-WT– GFP, HFE-N110A–GFP, HFE-N130A–GFP, HFE-N234A–GFP or HFE-NNN110/130/234AAA–GFP triple mutant Sixteen to eighteen hours post transfection the cells were immunostained with an anti-TfR1 Ig, and processed for fluorescence microscopy Scale bar, 10 lm (B) Graph showing the relative amounts of transfected cells exhibiting ERC/punctate or reticular localization of expressed HFE proteins (n = 3).

whether N-glycosylation is important for HFE

regula-tion of transferrin uptake To this end, transfected

HuTu80 cells expressing HA-tagged forms of wild-type

or mutant HFEs were analysed for their ability to bind

fluorescently labelled transferrin Consistent with

previ-ous reports, we noted that cells expressing wild-type HFE displayed a striking decrease in cell-surface bind-ing of transferrin, and that little or no reduction in transferrin binding was observed in cells expressing the HFE-C282Y mutant (Fig 7A)

A

B

Fig 5 Intracellular localization of HFE double N-glycosylation site mutants (A) HuTu80 cells were transfected to express HFE-NN110/130AA–HA, HFE-NN130/ 234AA–HA or HFE-NN234/110AA–HA Six-teen to eighSix-teen hours post transfection the cells were immunostained with an anti-TfR1

Ig, and processed for fluorescence micros-copy Scale bar, 10 lm (B) Graph showing the relative amounts of transfected cells exhibiting ERC/punctate or reticular localiza-tion of expressed HFE proteins (n = 4).

In experiments to compare the status of our

glyco-sylation mutants in this assay we observed that the

tri-ple mutant displayed a phenotype indistinguishable

from the C282Y mutant, with an almost complete loss

of the ability to regulate transferrin binding (Fig 7B)

By contrast, cells transfected with either the single or

double mutants were all capable of reducing

transfer-rin binding In each case, however, there tended to be

a decrease in the proportion of cells that retained

this ability compared with the wild-type protein

(Fig 7B–D) This effect was strongest for the 110/130

double mutant (Fig 7D)

Discussion

Although N-glycosylation can dramatically alter the

structure and function of many proteins, there are also

cited instances in whch the mutation of glycosylation sites has little or no effect [42,44] The importance of glycosylation is highly variable, therefore, and even in cases where it is important, the effect may be either direct or indirect In this study, we set out to explore the importance of HFE glycosylation In doing so, we aimed to expand on previous studies, which despite ref-erence to glycan addition and the development of endo-H resistance [13,18], nevertheless failed to identify the role, if any, that glycosylation plays in HFE locali-zation and function We established for the first time that the protein becomes N-glycosylated at asparagine residues 110, 130 and 234, and that mutation of all three sites results in the production of a protein that is glycosylation deficient Glycosylation at each of the substrate asparagine residues can occur independently

of the glycosylation status at the other two sites

A

B

Fig 6 ER localization of the HFE-triple mutant and interaction of N-glycosylation site mutants with b2M (A) HuTu80 cells transfected to express HFE-triple mutant–HA were immunostained with anti-HA and anti-BiP Ig, and processed for fluorescence microscopy Co-localization masks were created as described in Materials and methods Scale bar, 10 lm (B) Constructs expressing HFE-WT–HA, HFE-NNN110/130/ 234–HA (triple), HFE-N110A–HA, HFE-N130A–HA, HFE-N234A–HA or HFE-C282Y–HA proteins were transiently transfected into HEK293T cells Forty-eight hours after transfection cells were harvested HA-tagged proteins in cleared cell lysates were fractionated on 11% SDS/ PAGE for immunoblotting with a mouse HA Ig (upper) b2M and b2M-interacting proteins were immunoprecipitated using a rabbit anti-b2M Ig and precipitated proteins were fractionated by SDS/PAGE (11%) for detection of HA-tagged proteins by immunoblot using a mouse anti-HA Ig (lower) In a complementary series of experiments NiNTA–agarose was used to precipitate His-tagged versions of the WT, C282Y and triple-mutant HFE proteins from transfected HEK293T cells Immunoblots confirmed that only the wild-type HFE protein was capable of co-precipitating significant quantities of b2M (data not shown).

B

Fig 7 Regulation of transferrin binding by N-glycosylation site mutants HuTu80 were transfected to express (A) HFE-WT–HA or C282Y–HA, (B) triple mutant HFE-NNN110/130/234–HA (triple) or single mutants HFE-N110A–HA, HFE-N130A–HA and HFE-N234A–HA or (C) double mutants HFE-NN110/130AA–HA, HFE-NN234/110A–

HA or HFE-NN234/110AA–HA, as indicated Sixteen hours post transfection the cells were serum starved for 2 h followed by incubation with Alexa Fluor 594-bound Tfn for 1 h at 4 C Cells were immunostained with an anti-HA Ig and processed for fluo-rescence microscopy Scale bar, 10 lm (D) Graph showing the percentage of

transfect-ed cells with rtransfect-eductransfect-ed transferrin binding in response to the expression of various HFE proteins as indicated *P < 0.05, **P < 0.01

by Student’s unpaired t-test (n = 6).