Proc. Nati. Acad. Sci. USAVol. 87, pp. 26-30, January 1990Biochemistry

Albumin Redhill (-1 Arg, 320 Ala -- Thr): A glycoprotein variant ofhuman serum albumin whose precursor has an aberrant signalpeptidase cleavage siteSTEPHEN 0. BRENNAN*t, TIMOTHY MYLES*, ROBERT J. PEACH*, DAVID DONALDSONt,AND PETER M. GEORGE**Molecular Pathology Laboratory, Department of Clinical Biochemistry, Christchurch Hospital, Christchurch, New Zealand; and tDepartment of ChemicalPathology, East Surrey Hospital, Redhill, Surrey RH1 5RH, United Kingdom

Communicated by Frank W. Putnam, July 24, 1989 (received for review May 25, 1989)

ABSTRACT Albumin Redhill is an electrophoreticallyslow genetic variant of human serum albumin that does notbind 63Ni21 and has a molecular mass 2.5 kDa higher thannormal albumin. Its inability to bind Ni2+ was explained by thermding of an additional residue of Arg at position -1. This didnot explain the molecular basis of the genetic variation (sinceproalbumin contains adjacent Arg residues at -1 and -2) orthe increase in apparent molecular mass. Fractionation oftryptic digests on concanavalin A-Sepharose followed by pep-tide mapping ofthe bound and unbound fractions and sequenceanalysis of the glycopeptides identified a mutation of320 Ala-Thr. This introduces an Asn-Tyr-Thr oligosaccharide attach-ment sequence centered on Asn-318 and explains the increasein molecular mass. This, however, did not satisfactorily explainthe presence of the additional Arg residue at position -1. DNAsequencing of polymerase chain reaction-amplified genomicDNA encoding the prepro sequence of albumin indicated anadditional mutation of -2 Arg-- Cys. This introduces a preprosequence, Met-Lys-Trp-Val-Thr-Phe-Ile-Ser-Leu-Leu-Phe-Leu-Phe-Ser-Ser-Ala-Tyr-Ser-Arg-Gly-Val-Phe-Cys-Arg (cf.-Tyr-Ser-Arg-Gly-Val-Phe-Arg-Arg- in normal human pre-proalbumin). We propose that the new Phe-Cys-Arg sequencein the propeptide is an aberrant signal peptidase cleavage siteand that the signal peptidase cleaves the propeptide of albuminRedhill in the lumen of the endoplasmic reticulum before itreaches the Golgi vesicles, the site of the diarginyl-specificproalbumin convertase.

Human serum albumin is a 585-residue single-chain proteinmade up of three internally homologous domains (1). It issynthesized in the liver as preproalbumin, and the pre-(signal) peptide is cleaved off as the precursor protein entersthe lumen of the endoplasmic reticulum (2). As albumincontains no carbohydrate it undergoes no further modifica-tion until it reaches the Golgi vesicles, where a secondN-terminal modification occurs: cleavage that removes thepropeptide Arg-Gly-Val-Phe-Arg-Arg (3). The product, se-rum albumin with an N-terminal sequence of Asp-Ala-His-Lys-, is then constitutively secreted into circulation,where it has a half-life of 19 days (1).There have been a number of instances in which specific

point mutations have interfered with this processing (4).Proalbumin Christchurch (-1 Arg -> Gln) (5), proalbuminLille (-2 Arg -- His) (6), and proalbumin Blenheim (1 Asp -3

Val) have each played a crucial role in the identification oftheauthentic mammalian diarginyl-specific proalbumin "conver-tase" (4, 7, 8), which is a Ca2+-dependent protease (7).The report of albumin Redhill as a genetic variant with an

additional arginine residue at position -1 (9) prompted us to

ask the questions: what was the molecular basis of the geneticvariation, since proalbumin normally contains adjacent argi-nine residues at -1 and -2; and what could be learned fromthis case about the intracellular processing of albumin; inparticular, what did it mean in terms of the specificity of theproalbumin convertase.The original investigation of albumin Redhill reported its

occurrence in a mother and son of Anglo-Saxon origin andsuggested that, in addition to the presence of the N-terminalArg, there may also be an abnormality beyond the Trp atposition 214 (10). Here, we substantiate this finding and showthat albumin Redhill is a glycoprotein with an Asn-linkedoligosaccharide attached at position 318 consequential to themutation 320 Ala -+ Thr. Further, we show that the DNAencoding the precursor of albumin Redhill contains a muta-tion which indicates a substitution of -2 Arg -+ Cys: thisintroduces a spurious signal peptidase cleavage site.

MATERIALS AND METHODSMaterials. Sialidase type VI (Clostridium perfringens) was

obtained from Sigma. Endo-j3-N-acetylglucosaminidase F(Endo-F; Flavobacterium meningosepticum) and 63NiC12were obtained from New England Nuclear. Arg-albumin waspurified from the plasma of the boy with a1-antitrypsinPittsburgh (11). Proalbumin variants and antithrombin RouenIII, used here as markers, were previously purified andcharacterized in this laboratory (Christchurch).

Purification. Plasma was collected from the mother (theproposita), and 30 ml was dialyzed against 2 mM EDTA,freeze-dried, and delivered by airmail from Redhill, U.K., toChristchurch. On arrival, it was reconstituted with 30 ml ofwater. Albumin components were purified by DEAE-Sephadex chromatography using 16 mM acetate buffer and apH gradient from pH 5.2 to 4.5 (12). All fractions wereassayed by Laurell "rocket" electrophoresis into anti-albumin antisera (13).

Effect of Sialidase and Endo-F. Proteins were dissolved (2mg/ml) in 15 ,ul of 0.1 M sodium acetate buffer, pH 5.6/10mM CaC12. To this was added 1 ,ul of sialidase (1 mg/ml inwater) (14). Incubations were carried out at 20°C for up to 3hr, and the extent of reaction was assessed by agarose gelelectrophoresis at pH 8.6. Endo-F digestions were carried outat a substrate concentration of 1 mg/ml in 25 ,ul of 0.1 Msodium phosphate buffer, pH 6.1/50 mM EDTA/0.1% SDS/1% Nonidet P-40. Endo-F (0.05 unit) was added and incuba-tions were conducted for up to 24 hr at 20°C (15). The extentof reaction was assessed by SDS/PAGE.

Abbreviations: Endo-F, endo-,B-N-acetylglycosaminidase F; PCR,polymerase chain reaction; PTC, phenylthiocarbamoyl; PTH, phen-yrthiohydantoin.

tTlo whom reprint requests should be addressed.

26

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Proc. Natl. Acad. Sci. USA 87 (1990) 27

Gel Electrophoresis. Agarose gel electrophoresis was per-formed in 1% agarose in Tris-barbitone buffer (38 mMTris/46 mM sodium barbitone/16mM diethylbarbituric acid,pH 8.6) and 63Ni autoradiography was carried out as previ-ously described (16). SDS-PAGE was performed under re-ducing and nonreducing conditions in polyacrylamide gelgradients of 7.5-12.5% and 10-20o at pH 8.3 (17).

Protein Fragmentation. Limited tryptic digestion was car-ried out on native albumin; 100 ,.g was incubated with trypsin(2% wt/wt) for 30 min in 30 pl of 2 mM Tris HCI, pH 8.5 (18).All other fragmentation procedures were carried out onS-carboxamidomethylated albumin (reduced and derivatizedin 8 M urea), including partial acid hydrolysis (12), CNBrcleavage (19), and total tryptic digestion (12).

Peptide Purification. S-Carboxymethylated albumin (20mg) was digested with trypsin and applied to a column (1.6 x10 cm) of concanavalin A-Sepharose equilibrated in 25 mMNH4HC03/0.5 mM MnCI2/0.5 mM MgCl2/0.5 mM CaC12.Unbound peptides were directly eluted with this same buffer.Bound peptides were eluted with 25 mM NH4HCO3 contain-ing 30 mM methyl a-D-glucoside and freeze-dried (14). Themethyl a-D-glucoside was extracted with 80% (vol/vol) eth-anol. Bound and unbound peptide fractions were analyzed bytwo-dimensional peptide mapping (14, 20). Analytical mapswere stained with cadmium/ninhydrin (20) and overstainedwith a-nitroso-f8-naphthol (21). Preparative maps werestained with 0.002% fluorescamine and peptides were elutedwith 1% acetic acid (14).

Structural Analysis. Peptides were hydrolyzed in 6 M HCI(110°C, 18 hr). When nanomole quantities were available,compositions were determined on a Dionex D-300 amino acidanalyzer using ninhydrin detection. When lesser amountswere available, hydrolysates were derivatized with phenyl-isothiocyanate, and the resulting phenylthiocarbamoyl (PTC)amino acids were quantified by reverse-phase HPLC (22).Sequence analysis employed a manual Edman degradation

procedure and phenylthiohydantoin (PTH) amino acids wereidentified by reverse-phase HPLC (22). After each cycle, asample was removed for dansyl analysis and dansyl aminoacids were identified by chromatography on 2.5 x 2.5 cmpolyamide plates (23).Polymerase Chain Reaction (PCR) and DNA Analysis. Ge-

nomic DNA was extracted from whole blood by a phenol/chloroform method (24) and delivered to Christchurch byairmail. Four oligonucleotides were synthesized on an Ap-plied Biosystems DNA synthesizer and had the followingsequences: TM8, 5'-ATGTGCAGTTTCCCTCCGTTT-GTCCTAGCT-3'; TM9, 5'-ATGGATCCCTTTGCACTT-TCCTTAGTGCGC-3'; TM11, 5'-CCTAGCTTTTCTCT-TCT-3'; and TM12, 5'-GCATGTCGACAAAACACACCC-3'. (Underlined sequences are introduced Pst I and BamHIrestriction sites.) A 400-base-pair fragment of the genomicDNA encoding the first exon of the albumin gene (25) (aminoacids -24 to +2) was amplified by PCR (26) using TM8 andTM9 as primers. The amplification used heat-stable TaqDNA polymerase C (Perkin-Elmer/Cetus) and 30 cycles ofdenaturation (93°C, 60 s), annealing (65°C, 60 s), and exten-sion (73°C, 90 s) with a final extension phase of 10 min. The400-base-pair PCR product was gel-purified and sequenced,using 32P-endlabeled TM11 as the sequencing primer andSequenase 2.0 (United States Biochemical) in a standardprotocol (27, 28).

In addition, PCR products were electrophoresed in a 1%agarose gel and transferred to GeneScreenPlus by alkalinetransfer (29). The membrane was hybridized at 50°C to32P-end-labeled TM12 (specific activity 109 cpm/,ug) in 6xSSC/0.1% SDS containing denatured herring sperm DNA at0.2 mg/ml (lx SSC is 0.15 M NaCI/0.015 M sodium citrate,pH 7.0). After hybridization, the filter was washed twice at650C in 6x SSC/0.1% SDS and autoradiographed.

RESULTSAgarose gel electrophoresis of the proposita's plasmashowed that the variant was more positively charged thannormal albumin and that it did not bind 63Ni (Fig. 1). Since therequirement for Cu2+ or Ni2' binding is a free a-amino groupon residue 1 and a histidine in position 3 (16), this wasconsistent with the earlier finding that albumin Redhill con-tained an additional arginine residue at position -1 (9);however, the variant ran more cathodally than Arg-albumin(Fig. 1, lane 3). Densitometric scanning ofthe electrophoreticplates, integration of the DEAE-Sephadex elution profile,and gravimetric analysis of the recovered albumins (below)all indicated that the variant made up approximately 37% ofthe total plasma albumin.A surprising result was found when plasma was chromato-

graphed on DEAE-Sephadex. Despite the additional argi-nine, albumin Redhill eluted after the normal albumin com-ponent. Indeed, we had previously shown that authenticArg-albumin elutes ahead of albumin under these conditions(30). Nevertheless, both albumin components were collectedand found to be about 95% pure (Fig. 1, lanes 5 and 6).

Analysis of the normal component gave the expected initialsequence, Asp-Ala-His-Lys-. The initial sequence ofalbuminRedhill was confirmed as being Arg-Asp-Ala-His-Lys-. SDS/PAGE, however, confirmed that albumin Redhill was notmerely Arg-albumin: it had an apparent molecular mass about2.5 kDa higher than normal albumin and Arg-albumin (forexample, see Fig. 3, lanes 6, 9, and 12).The anomalous elution from DEAE-Sephadex and the

apparent increase in molecular mass could be accounted forby the presence of a complex oligosaccharide. Such a struc-ture would have a molecular mass ofabout 2.5 kDa and wouldterminate in sialic acid residues. To test this possibility, thenormal and variant albumins, together with a control ofantithrombin, were incubated with sialidase (Fig. 2). Over a3-hr incubation, antithrombin loses eight residues of sialicacid and moves to the cathode on electrophoresis (14). Asexpected, the normal albumin was unaffected by this treat-ment. Albumin Redhill, however, shifted to the cathode,suggesting that it contained an oligosaccharide moiety.The oligosaccharide of albumin Redhill was resistant to

Endo-F cleavage even though incubations were conductedunder denaturing conditions (0.1% SDS/1% Nonidet P-40)for 24 hr (Fig. 3). Failure of cleavage could not be attributedto inappropriate conditions, since in the same experimentantithrombin yielded the expected ladder of four bandscorresponding to the successive removal of its four N-linkedoligosaccharides. Lanes 4 and 5 are markers of normalantithrombin and a genetic variant, antithrombin Rouen III (7Ile Asn), which contains an additional N-linked oligosac-

1 2 3 4 5 6

FIG. 1. Agarose gel electrophoresis (pH 8.6) showing plasmacontrols and purified albumins. Lanes: 1, plasma from heterozygouscarrier of proalbumin Blenheim (1 Asp -* Val); 2, plasma from carrierof proalbumin Christchurch (-1 Arg -. Gin); 3, Arg-albumin; 4,plasma from proposita, 5, purified normal albumin from proposita; 6,purified albumin Redhill. Prior to electrophoresis 0.2 AGCi (1 Ci = 37GBq) of 63Ni2+ was added to each sample. The 63Ni2' autoradio-graph is shown above the Coomassie blue-stained pattern.

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28 Biochemistry: Brennan et al.

- m

1 2 3 4 5 6 7 8 9

FIG. 2. Agarose gel electrophoresis (pH 8.6) showing effect ofsialidase. Lanes 1-3, antithrombin control incubated with sialidasefor 0, 1, and 3 hr, respectively. Lanes 4-6, albumin Redhill incubatedfor 0, 1, and 3 hr, respectively. Lanes 7-9, normal albumin compo-nent incubated for 0, 1, and 3 hr, respectively.

charide on its new Asn residue (15). It is pertinent to note thatunder reducing conditions, normal albumin runs in the same

position as normal antithrombin, while albumin Redhill comi-grates with antithrombin Rouen III.To locate the attachment site of the putative oligosaccha-

ride, an albumin control, together with the normal and variantalbumin from the proposita, was subjected to a series ofproteolytic cleavages designed to yield large fragments.

Partial acid hydrolysis cleaves S-carboxymethylated hu-man albumin at its single Asp-Pro bond between residues 365and 366 to give a 43-kDa N-terminal component and a 25-kDaC-terminal component (12). While normal albumin gave theseexpected bands on SDS/PAGE, the N-terminal fragment ofalbumin Redhill had a higher apparent molecular mass (Fig.4, lanes 4-6).

Limited tryptic digestion of native albumin cleaves atArg-197 to give a stable 43-kDa fragment consisting ofdomains 2 and 3 (18). The N-terminal domain is degradedmore extensively by trypsin and is not detected. Limitedtryptic digestion of normal albumin generated the expected43-kDa band. This band, however, had a higher apparentmolecular mass in the case of albumin Redhill (Fig. 4, lane13). This finding, with the results from the partial acidhydrolysis, places the abnormality in the second domain,between residues 198 and 365.There was no easily discernible difference between CNBr

digests (Fig. 4, lanes 7-9). This places the mutation in one ofthe small (<4-kDa) CNBr fragments. Only one such fragmentarises from the second domain, implying that the mutationmust be between Met residues 298 and 329.To confirm the existence of a carbohydrate side chain and

to isolate possible glycopeptides, S-carboxymethyl deriva-tives of the normal and variant albumin were subjected tototal tryptic digestion and fractionated on concanavalin A-Sepharose. In both cases the unbound peptides were eluteddirectly in the equilibration buffer and mapped (Fig. 5). Anybound peptides were eluted with 30 mM methyl a-D-glu-coside and mapped in a similar manner (Fig. 5). As albuminis not a glycoprotein, no peptides were recovered from the"bound" fraction of the normal component. The boundfraction from albumin Redhill, however, contained twohighly polar negatively charged tryptic peptides (Rhl andRh2). Rh2 but not Rhl stained yellow with Cd/ninhydrin (20),indicating they contained different N-terminal residues. Bothpeptides contained tyrosine as indicated by overstaining.PTC amino acid analysis (Table 1) ofpeptides isolated from

fluorescamine stained maps showed that Rhl contained twoLys residues and spanned residues 314-323, while Rh2 was

a shorter version of this same peptide, spanning residues318-323. One of the two Ala residues in each of thesepeptides, however, was missing and replaced by Thr; this

-- -

q

1 2 3 4 5 6 7 8 9 10 11 12

FIG. 3. SDS/PAGE showing the effect of Endo-F. Lanes 1-3,antithrombin control incubated for 0, 6, and 24 hr, respectively, withEndo-F. Lanes 4 and 5, markers of antithrombin and antithrombinRouen III (7 Ile -+ Asn). Lanes 6-8, normal albumin from propositaincubated with Endo-F for 0, 6, and 24 hr, respectively. Lanes 9-11,albumin Redhill incubated for 0, 6, and 24 hr, respectively, withEndo-F. Lane 12, marker of Arg-albumin. The gel contained a7.5-12% acrylamide gradient and was run under nonreducing con-ditions.

being so, it would be expected that the unbound peptidefraction from albumin Redhill would lack the predictedneutral tryptic peptide, Asn-Tyr-Ala-Glu-Ala-Lys. Whenmaps of the unbound fractions were overstained with a-nitroso-,-naphthol, it was apparent that a neutral tyrosine-containing peptide (A2) was indeed missing from the unboundfraction of albumin Redhill (Fig. 5).

It appeared, therefore, that the aberrant peptide Rhlresults from partial tryptic cleavage of peptide Rh2 at Lys-317. To obtain a single peptide for sequence analysis, theexperiment was repeated, allowing 48 hr for tryptic digestioninstead of 3 hr. Maps of the bound fraction from albuminRedhill then showed a single peptide which corresponded topeptide Rh2. Amino acid analysis, this time with ninhydrindetection, confirmed a composition encompassing residues318-323 but with one of the two residues of Ala replaced byThr. With ninhydrin detection it was also possible to dem-onstrate that peptide Rhl contained four residues of gluco-samine, the hydrolysis product of N-acetylglucosamine.To determine whether the oligosaccharide side chain was

attached to Asn-318 or to the new Thr, it was important toidentify the glycated residue. For this reason samples weretaken for dansyl analysis at the end of each Edman cycle as

im *

_- _

1 2 3 4 5 6 7 8 9 10 11 12 13 14

FIG. 4. SDS/PAGE showing fragmentation pattern of albuminRedhill and the localization of the mutation site. Lanes 1-3, S-carboxamidomethyl derivatives of albumin control, normal albuminfrom proposita, and albumin Redhill, respectively. Lanes 4-6, partialacid hydrolysis products of normal albumin, normal albumin fromproposita, and albumin Redhill, respectively. Lanes 7-9, CNBrcleavage products of normal albumin, normal albumin from propos-ita, and albumin Redhill, respectively. Lanes 10 and 11, nativenormal and variant albumin from proposita. Lanes 12 and 13, limitedtryptic cleavage fragments of normal and variant albumins, respec-tively. Lane 14, plasma from proposita. The gel contained a 10-20%oacrylamide gradient and was run under nonreducing conditions.

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*

*"- 4w_

_ A_

+

Rhl Rh2

+FIG. 5. Peptide maps of the bound and unbound tryptic peptides

from concanavalin A-Sepharose columns. The upper pair showunbound fraction from albumin Redhill and normal albumin, respec-tively. Peptide A2 is missing from the unbound fraction of albuminRedhill. The lower pair of maps shows the bound peptide fractionfrom normal albumin and albumin Redhill, respectively. AlbuminRedhill contains two glycopeptides, Rhl and Rh2. Electrophoresiswas at pH 6.5 and chromatography was conducted in the upper phasefrom pyridine/isoamyl alcohol/water (6:6:7, vol/vol).

well as performing the usual PTH identification. No N-terminal residue could be detected by using PTH analysis.This procedure established a sequence of Xaa-Tyr-Thr-Glu-Ala-Lys for peptide Rh2. The hydrolytic conditionsof the dansyl procedure gave a sequence of Asx-Tyr-Thr-Glx-Ala-Lys. This establishes a substitution of 320 Ala

Thr in albumin Redhill, and this mutation introduces aAsn-Tyr-Thr glycosylation sequence centered on Asn-318.To determine why albumin Redhill contained an additional

residue of Arg at position -1, genomic DNA was amplified

Table 1. Amino acid analysis of glycopeptides fromalbumin Redhill

No. of residues per peptide

PTC Ninhydrin Expected

Residue Rhl Rh2 Rh2 318-323 314-323Asp 1.7 0.9 1.0 1 2Glu 1.0 1.1 1.2 1 1Cys* 0.8 - 1Thr 0.9 0.7 1.0 0 0Ala 0.9 1.1 1.0 2 2Tyr 1.2 1.1 0.9 1 1Val 0.8 - 1Lys 1.8 0.8 0.9 1 2GlcNt 3.8 0 0*As carboxymethylcysteine.tThe GlcN standard was subjected to a mock hydrolysis togetherwith amino acid standards.

FIG. 6. (A) DNA sequencing gel showing preproalbumin se-quence of albumin Redhill. Arrow indicates the new thymidine basedue to mutation of C to T at codon for residue -2. (B) Agarose gelelectrophoresis showing PCR amplification products. Lanes 1 and 2,normal controls; lanes 3-6, proposita; lane 7, markers (Hae Ill digestof 4X174 DNA). (C) Autoradiograph of B probed with oligonucle-otide TM12, which encodes Cys at position -2.

by using the PCR procedure. The primers TM8 and TM9 wereused to amplify a 400-base-pair sequence encoding the firstexon of the albumin gene, amino acid residues -24 to +2.This includes the signal peptide (-24 to -7), the propeptide(-6 to -1), and the first two residues of mature albumin.Sequence analysis of the amplified DNA (27, 28) gave theexpected sequence up to the TTT coding for 'the Phe atposition -3 of the propeptide (Fig. 6A). Two forms of thenext codon were present, CGT and TGT, the former codingfor the expected Arg but the latter coding for a Cys at position-2. These data indicate that the proposita is heterozygous fora mutation of -2 Arg -* Cys. This finding was confirmedindependently by the specific hybridization of TM12 (anoligonucleotide encoding -2 Cys) to DNA from the propositabut not to DNA from the controls (Fig. 6 B and C).

DISCUSSIONThe original description of albumin Redhill as an inheritedvariant of albumin (9, 10) is explaine4 by the finding here ofa mutation of 320 Ala -+ Thr, and the molecular mass increaseis explained by the presence of a complex oligosaccharideattached to Asn-318. It is not clear Why this N-linked oligo-saccharide is resistant to Endo-F cleavage, but differentglycoproteins are known to exhibit extreme variability intheir susceptibility to hydrolysis by Endo-F (31).Albumin Redhill is the only instance we know of in which

serum albumin occurs as a glycoprotein, and the molecularmass, increase of 2.5 kDa could fit either a biantennary(2222-Da) or a triantennary (3030-Da) structure. While adefinitive assessment of the oligosaccharide structure mustawait further analysis, the presence of both N-acetylglu-cosamine and sialic acid is indicative of a fully matureoligosaccharide typical of those in other plasma glycopro-teips exported from the liver (32).Albumin Redhill elutes from DEAE-Sephadex (pH 4.5) in

the same position as albumin Canterbury (313 Lys -* Asn)(22), which contains two net negative charges more thanArg-albumin and one more than normal'albumin. This isconsistent with the presence of the new Arg and two residuesof sialic acid. Its electrophoretic mobility at'pH 8.6, runningbetween Arg-albumin and proalbumin Christchurch, is puz-zling. Indeed, asialo-Redhill (+1) runs more cathodally thanproalbumin Christchurch (+2). This, presumably, resultsfrom perturbation of secondary and tertiary structure by theoligosaccharide which may alter the external charge distri-bution on the putative helix to which it is attached. Pertur-bation of helix structure is a common explanation for sup-pression of charge changes in hemoglobin variants (33).The presence of the new Arg at position -1 appears to be

unrelated to the 320 Ala -* Thr mutation and possible stericconstraints the new oligosaccharide may place on precursor

BA

ACG T

A2

:1Iom..A2 --m~.o .....p-,

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30 Biochemistry: Brennan et al.

24 pre -7 pro -1

Preproalbumin (normal) M K W V T F I S L L F L F S S A Y S R G V F R R

Preproalbunin Redhill M K W V T F I S L L F L F S S A Y S R G V F C R

Proalbumin Christchurch R G V F R Q

Proalbumnin Lille R G V F H R

FIG. 7. Predicted sequence of preproalbumin Redhill based on DNA sequence. Signal peptidase cleavage site and proposed new cleavagesite are underlined. Proalbumin convertase cleavage site is doubly underlined.

processing. Sequence analysis of PCR-amplified genomicDNA indicated a heterozygous state for a mutation of -2 Arg

Cys. This mutation alone is sufficient to account for thepresence of the additional Arg at position -1. The predictedamino acid sequence of preproalbumin Redhill is shown inFig. 7. The -2 Arg -- Cys mutation in the propeptide ofalbumin Redhill (R G V F C R) results in the loss of thediarginyl proalbumin cleavage site. Initially, this result ap-peared enigmatic, as the genetic variants proalbumin Christ-church (-1 Arg-- Gln) and proalbumin Lille (-2 Arg-+ His)also have mutations in this diarginyl propeptide cleavage site,and these are exported from the liver as their respectiveproalbumins, not as Arg-albumin. The proalbumin conver-tase is a Ca2+-dependent membrane-bound enzyme located inGolgi vesicles (3, 4, 7), and in vitro experiments show thatdetergent-solubilized extracts of these vesicles, while capa-ble of cleaving normal human proalbumin, fail to cleavevariants that lack the diarginyl site (4, 7). Proalbumin Redhill(-2 Arg-+ Cys) should not be a substrate for this enzyme andshould (if it existed in the first place) be exported as proal-bumin Redhill. We propose that the export intermediateproalbumin Redhill is never formed and that the only intra-cellular cleavage is by the signal peptidase located in theendoplasmic reticulum. The new Phe-CyssArg sequence isvery similar to the signal peptidase cleavage site sequenceTyr-Ser1Arg and, indeed, the signal peptidase cleavage site inrat preproalbumin is Phe-Ser'Arg- (34).The amino acid immediately N-terminal to the signal

peptidase cleavage site is the most critical in determiningwhere the enzyme cleaves and, indeed, cleavage can occur atmultiple sites if alternate spurious sites are available (35, 36).In the Redhill case two sites are available, the "correct"Tyr-Ser'Arg site and the new Phe-Cys'Arg site. Because noproalbumin Redhill was detected in circulation, we suggestthat the new site is used in preference to the "correct" site,giving rise to Arg-albumin Redhill. Site-directed mutagenesisexperiments with pre(Apro)apolipoprotein A-ll have, in fact,shown that Cys is favored over Ser in directing high-fidelitycleavage by signal peptidase (36).The data presented here explain the initial observations of

the Redhill case (10) and confirm the suggestion of twomutations. Because of the extraordinary finding of twoindependent mutations, both affecting the intracellular pro-cessing of albumin, DNA sequencing was repeated in fouramplification experiments with the same results. This -2 Arg-- Cys substitution was confirmed by the hybridization ofDNA to the oligonucleotide probe encoding a Cys at -2.There may be other instances of mutation at residue -2; six

unrelated cases of circulating Arg-albumin have been broughtto our attention (personal communications, Frank W. Put-nam and Carl-Bertil Laurell). While the reason for thepresence of the additional Arg has not yet been established inthese cases, it was clear that a new oligosaccharide side chain

was not involved. It may be that the -2 Arg -+ Cys mutationrepresents a (very) minor polymorphism and that the 320 Ala

Thr substitution has occurred in this allele.

This investigation was supported by the Medical Research Councilof New Zealand.

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2. Peters, T., Jr. (1987) Clin. Chem. 33, 1317-1325.3. Judah, J. D. & Quinn, P. S. (1978) Nature (London) 271, 384-385.4. Brennan, S. 0. (1989) Mol. Biol. Med. 6, 87-92.5. Brennan, S. 0. & Carrell, R. W. (1978) Nature (London) 274, 908-909.6. Abdo, V., Rousseux, J. & Dautrevaux, M. (1981) FEBS Lett. 131,

286-288.7. Brennan, S. 0. & Peach, R. J. (1988) FEBS Lett. 229, 167-170.8. Bathurst, I. C., Brennan, S. O., Carrell, R. W., Cousens, L. S., Brake,

A. J. & Barr, P. J. (1987) Science 235, 348-350.9. Hutchinson, D. W. & Matejtschuk, P. (1985) FEBS Lett. 193, 211-212.

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