Biochimie (1991) 73,617--6!0

617

© Soci6t6 franqaise de biochimie et biologie molrculaire / Elsevier, Paris

Short communication

A l b u m i n Paris 2: a new genetic variant distinguished by isoelectric focusing D Rochu, H Crespeau, JM Fine Laboratoire d' lmmu~ochimie Analytique, lnstitut National de Transfusion Sanguine, 6 rue Alexandre Cabanel. 75739 Paris Cedex 15, France

(Received 14 December 1990; accepted 25 January 1991)

Summary - - Until recently, the characterization of genetic variants of human serum albumin was performed by electrophoretic typing prior to the determination of their amino acid substitutions. We describe a procedure using isoelectric focusing in the presence of urea for the analysis of the genetic variation of albumin. This procedure allowed a clear distinction of a new variant, previously found to be identical with albumin Sondrio according to its relative electrophoretic mobilities at 3 pHs. This new variant, the third rare albumin allotype identified in the Ile-de-France region, was called albumin Pads 2. albumin / genetic variant / isoelectric focusing

Introduction

Although present methodologies allow examination of the p o l y m o r p h i s m of proteins at the genomic level, data concerning the variability of human serum albumin have mostly been provided by studies carded out at the protein level. Indeed, analysis of restriction fragment length has evidenced intronic mutations only [ 1, 2], and discrepancies observed in c D N A sequences probably arise from artifacts in cloning or in c D N A sequence analysis [3]. In contrast, alloalbuminemia is detectable by electrophoresis and more than 100 alloalbumins have been identified by electrophoresis connected with clinical study or blood donnor screening [4,5]. A first attempt at classification was performed by Weitkamp in 1973 [6, 7]. Since then, a new technique has been developed for an accurate estimation of the relative mobilities of the variants at 3 pHs allowing reproducible typing [5, 8]. On the other hand, isoelectric (IEF) is generally very efficient for studying polymorphism of various human proteins. Meanwhile, it is worth noting that little work in IEF of albumin has been reported. This m a y be explained by the difficulty in obtaining a small number of sharply focused bands. On the basis of these data, we have developed a new procedure to investigate by IEF the behavior of albumin variants in addition to the results of the

conventionally employed electrophoretic typing. The purpose of this paper is to show that IEF allows genetic variants of human albumin to be distinguished which were not differentiated by electrophoresis and which therefore could have been considered as known allotypes. Materials and Methods

The serum of a hemophilic patient (RIG) from the Ile-deFrance region exhibited a bisalbuminemic pattern detected in the course of routine examination by electrophoresis. Identification of the albumin variant, using the determination of relative mobilities at 3 different pHs [5, 8] provided values identical to that of albumin Sondrio. IEF was carried out in horizontal polyacrylamide slab gel (258 x 124 x 0.5 mm) containing carder ampholyles in the pH range 5-8, in the presence of 8 M urea as previously described [9]. Albumin fractions containing both normal and albumin variants (Sond io or RIG) were purified by affinity chromatography on Blue-Trisacryl. Results and Discussion

Purified normal albumin exhibited heterogeneity in IEF and could be resolved into 2 major bands with different isoelectric points. Compared to this simple pattern, purified albumin fractions from heterozygous genetic alloalbuminemia provided more complex

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patterns with additional bands due to the presence of the variant albumin [9]. Tyro groups of variants could be evidenced according to the position of additional bands, either with more anodal or cathodal pls. The patterns obtained were consistent with the expected incidence in structural change of the variant, responsible for the modified net electric charge. For this reason, we studied by IEF the reference samples available in our laboratory, corresponding to different variants observed in Europe and which have been previously subjected to characterization by their amino acid substitution [9, 10]. A French reference sample (RIG) was previously identified as a Sondrio variant [11], according to its relative electrophoretic mobilities at 3 pHs, compared to that of the Italian reference sample (SO/BS) containing albumin Sondrio. Table I presents the results of the estimated relative mobilities of the variant (RIG) which suggested that it be classified as a Sondrio type. The recent determination of the structural change responsible for albumin Sondrio (333 Glu ---> Lys) [12], together with our observation that the same substitution, arising at different locations, such as in albumin Roma (321 Glu ---> Lys) [13], in albumin B (570 Glu ---> Lys) [14], or in albumin Vancouver (501 Glu -> Lys) [15] exhibit similar band patterns in 1EF, provided arguments for a new characterization of our sample. Figure 1 shows that the IEF banding pattern of the French sample (RIG) was not compatible with a Glu ---> Lys substitution, whereas the original Sondrio sample (SO/BS) exhibited a pattern in agreement with this type of structural change. These results demonstrate that the sample under study contained a new variant referred to as albumin Paris 2, evidenced by IEF while it was indistinguishable from albumin Sondrio by conventional electrophoresis, up to now the reference method used for identifying albumin variants prior to the biochemical characterization of their amino acid substitutions. After albumin Vanves [ 16] and albumin Paris [ 17], the latter exhibiting electrophoretic mobilities slower than that of Sondrio [7], albumin Paris 2 represents the third allotype in the Ile-de-France region. Taking this and the results presented here into account, it appears that IEF is likely to have an application in analyzing genetic variation of human albumin since it

+ 1

2

Fig 1. Characterization by IEF of albumin Pads 2. 1. Albumin Pads 2 (sample RIG); 2. albumin Sondrio (sample SO/BS).

is able to depict pl differences which are not sufficiently discernible by electi-ophoresis, even when conducted at v a r i o ~ nl-l~ "1~, etnnr,tHral ,~n~l,,o;o ~g j~

aa~,

L.VW.A W , ~ , L ~ t l

It~A

U, ltll[,IdtJ

t.~l~

V~JPl

this new variant by determination o f its amino acid

substitution is now in progress and will provide the final criterion for the complete characterization of albumin Paris 2.

Acknowledgments We are indebted to FW Putnam, F Porta, MF Ferrer-Le-Coeur and P Hivert, who kindly provided albumins Vancouver, Sondrio and Paris 2, respectively.

Table I. Electrophoretic typing of the albumin variant (RIG) by estimation of relative mobilities at 3 pHs. Samples

Relative mobilities pH 8.6

pH 6.9

pH 5.0

Albumin Sondrio

0.90 _+0.2

0.85 +_0.4

0.83 _+0.2

Albumin RIG

0.90

0.83

0.81

References 1 Murray J, Demopoulos CM, Lawn RM, Motulsky AG (1983) Molecular genetics of human serum albumin: restriction enzyme fragment length polymorphisms and analbuminemia. Proc Natl Acad Sci USA 80, 5951-5955 2 Lavaredade Souza S, Frain M, Momet E, Sala-Treoat JM, Lucotte G (1984) Polymorphisms in human alburn]n gene after DNA restriction by Hae III endonuclease. Hum Genet 67, 48-51

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9 10

Takahashi N, Takahashi Y, Blumberg BS, Putnam FW (1987) Amino acid substitutions in genetic variants of human serum albumin and in sequences inferred from molecular cloning. Proc Nail Acad Sci USA 84, 4413-4417 Tamoky AL (1980) Genetic and drug-induced variation in serum albumin. Adv Clin Chem 21,101-146 Fine JM, Marneux M, Rochu D (1987) Human albumin genetic variants: an attempt at a classification of european allotypes. Am J Hum Genet 40, 278-286 Weitkamp LR, Salzano FM, Neel JV, Porta F, Geerdink RA, Tarnoky AL (1973) Human serum albumin: twenty-three genetic variants and their population distribution. Ann Hum Genet 36, 381-392 Weitkamp LR, Mc Dermid EM, Neel JV, Fine JM, Petrini C, Bonazzi L, Ortali V, Porta F, Tanis R, Harris DJ, Peters T, Ruffini G, Johnston E (1973) Additional data on the population distribution of human serum albumin genes: three new variants. Ann Hum Genet 37, 217-226 Fine JM, Marneux M, Lambin P (1982) Human albumin variants: nomenclature of allotypes observed in Europe and quantitative estimation of their relative mobilities. Blood Trans Immunohematol 25, 149-163 Rochu D, Crespeau H, Fine JM (1991) Caract6risation des variants g6n6tiques de l'albumine humaine par focalisation iso61ectrique. Rev Fr Trans Hdmobio134, 35-47 Rochu D, Fine JM, Putnam FW (1988) Variants g6n6tiques de l'albumine humaine: caract6risation structu-

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rale des allotypes utilisds comme rdf6rences dans la classification 61ectrophor6tique. Rev Fr Trans lrnmnohdmatol 31,725-733 Porta F, Ruffini G, Pasino M, Scherini A (1971) Bisalbuminemia (aUoalbuminemia) of the slow type in an italian family. Exp Med In Congr Ser 233, 13 Galliano M, Minchiotti L, Madison J, Pumam FW, Watkins S, Rossi A, Porta F (1990) Caratterizzazione strutturale di alloa!bumine italiane. Bol Osp Varese 19, 211 Galliano M, Minchio~i L, Iadarola P, Ferri G, Zapponi MC, Castellani AA (1988) The amino acid substitution in albumin Roma: 321 Glu ---> Lys. FEBS Lett 233, 100104 Winter WP, Weikamp LR, Rucknagel DL (1972) Amino acid substitution in two identical inherited human serum albumin variants: albumin Oliphant and albumin Ann Arbor. Biochemistry 11,889-896 Huss K, Madison J, Ishioka N, Takahashi N, Arai K, Putnam FW (1988) The same substitution, glutamic acid ---> lysine at position 501, occurs in three alloalbumins of Asiatic origin: albumins Vancouver, Birmingham and Adana. Proc Nail Acad Sci USA 85, 66926693 Fine JM, Lambin P (1982) Albumin Vanves: a new fast moving variant of European origin. Hum Hered 32, 435-437 Sandor G, Martin L, Porsin M, Rousseau A, Martin M (1965) A new bisalbuminaemic family. Nature 208, 1222