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  • Blood, Vol. 59. No. 4 (April), 1982 747

    The Biochemical and Genetic Basis for the Microheterogeneity of Human R-Type Vitamin B12 Binding Proteins

    By Soo Young Yang, Peter S. Coleman, and Bo Dupont

    R-type vitamin B12 binding proteins (R proteins) from

    human granulocytes. erythrocytes. plasma, and other body

    fluids were characterized by isoprotein banding patterns

    on autoradiograms after resolution via thin-layer polyacryl-

    amide isoelectric focusing (IEF) gel electrophoresis. R pro-

    teins obtained from various tissue’ sources in a given

    individual show tissue-specific electrophoretic patterns.

    The desialated R proteins obtained following in vitro treat-

    ment with neuraminidase are. however, the same for any

    given individual and do not show tissue specificity. The

    differences seen in native R proteins (i.e.. transcobalamin I,

    Ill. and others) obtained from different tissues are due to

    V ITAMIN B,2 (COBALAMIN) is rarely found in the free state in the body, but is usually attached

    to specific proteins. In the review by Allen,’ vitamin

    B,2 binding proteins were classified according to their

    structural and functional properties: intrinsic factor

    (IF) in gastric juice mediates the absorption of the

    vitamin B,2 from the gastrointestinal tract; transcobal-

    amin II (TC II) occurs only in the plasma and facili-

    tates transport of the vitamin from blood to various

    tissues; and finally, the R proteins occur in most body

    fluids, including the plasma, and can also be found in

    some cells. The plasma contains two R proteins known

    as transcobalamin I (TC I) and transcobalamin III

    (TC III). The term “R protein” was originally devised

    by Simons and Gr#{228}sbeck2 to distinguish vitamin B,2

    binding proteins from IF in human gastricjuice. It was

    termed protein “R” because of its rapid electropho-

    retic mobility. This term is now used to denote cobal-

    amin-binding proteins from various sources such as

    saliva, leukocytes, milk, plasma, and amniotic fluids.

    These proteins are immunologically identical even

    though they may differ in molecular weight, electro-

    phoretic mobility, and carbohydrate content.’3 Be-

    cause many features are common among the R pro-

    teins from various tissues, Stenman4’5 introduced the

    term “cobalophilin” for the R proteins.

    Two fractions of R proteins in plasma can be

    distinguished by DEAE cellulose chromatography.

    One binds strongly to DEAE cellulose and has been

    termed TC I. The other, which binds only weakly to

    DEAE, is called TC IIl.68 TC I is mostly saturated

    with endogenous B,2 in normal plasma.913 In vivo, TC

    III normally is not found to any significant extent in

    plasma (the half-life in vivo is less than 5 mm).”3 But in

    vitro, unsaturated TC III is released from granulo-

    cytes, and this release can be inhibited by fluoride

    ion.9”4”5 The biochemical and genetic characteristics

    of TC II have recently been resolved.’�’8 Based on

    variations only in the sialic acid content. Granulocytes from

    patients with chronic myelogenous leukemia (CML) contain

    both TC I and TC Ill. and these R proteins can be released in

    vitro by lithium stimulation. Normal granulocytes contain

    only TC Ill. Differences in desialated R proteins from

    individual to individual are due to a genetic polymorphism

    controlled by a single genetic locus (designated TCR) with

    two alleles, 1 and 2. which are found to be codominantly

    expressed in heterozygous individuals. The allelic variants

    of the desialated R proteins found in different blood cells

    and body fluids are controlled by only one genetic locus.

    immunologic and biochemical similarities between R

    proteins from different tissues, it has been speculated

    that these proteins have a common phylogenetic origin

    and could be controlled by one genetic locus.45’92’

    Recently, Azen and Denniston22 have described a

    genetic polymorphism of R proteins of saliva. These

    authors demonstrated that the banding patterns of

    neuraminidase-treated samples from saliva, tears,

    milk, and leukocytes in isoelectric focusing electropho-

    resis were similar, but not identical, and could be

    explained by an autosomal inheritance of two codomi-

    nant alleles of one genetic locus. These previous studies

    did not make it possible to explain the relationship

    between TC I, TC Ill, and other R proteins and to

    explain the biochemical nature of the microhetero-

    geneity of these proteins.

    The present study demonstrates that TC I, TC III,

    and other native R proteins found in different normal

    tissues have different banding patterns on autoradio-

    grams of isoelectric focusing (IEF) gels. These differ-

    ences are tissue specific and caused by different degrees of sialation. Our study also demonstrated that

    leukemic granulocytes contain both TC I and TC III

    and that both these proteins can be released in vitro

    from the leukemic cells by lithium stimulation. Nor-

    mal granulocytes contain only TC III. Desialated R

    From the Human Immunogenetic Section, Memorial Sloan Ket-

    tering Cancer Center, and the Laboratory of Biochemistry, Depart-

    ment ofBiology, New York University. New York, N.Y.

    Supported in part by grants from the U.S. Public Health

    Services, National Institutes ofHealth, NCJ-CA 22507, CA 08748,

    CA 19267. and HD 15084.

    Submitted August 25, 1981; accepted December 1, 1981.

    Address reprint requests to Soo Young Yang. Ph.D.. Department

    of Immunogenetics, Sydney Farber Cancer Research Institute, 44

    Binney Street, Boston, Mass. 02115.

    © I 982 by Grune & Stratton, inc.


    For personal use only.on May 4, 2016. by guest www.bloodjournal.orgFrom

  • proteins, however, all have identical IEF banding

    patterns for a given individual, and this uniform IEF

    pattern is independent of the tissue of origin. Each

    individual’s R protein banding pattern is determined

    by a single autosomal genetic locus with two codomi-

    nantly expressed alleles.





    Plasma and Serum Collection

    Plasma and serum from venous blood were drawn into glass tubes

    (Vacutainer tubes, Becton-Dickinson, Rutherford, N i). Serum

    samples were obtained by allowing clot formation at room tempera-

    ture for 30 mm, followed by retraction of the coagel after I hr at 4#{176}C.

    The serum was separated by centrifugation at 4#{176}C,and divided into

    aliquots that were stored at - 70#{176}Cuntil used. Plasma was obtained

    by collecting blood samples in Vacutainer tubes (Becton-Dickinson)

    containing 10.5 mg of Na2EDTA. Plasma was separated immedi-

    ately after collection by centrifugation at 4#{176}C,divided into aliquots,

    and stored at -#{149}70#{176}C. Erythrocyte Lysates

    One milliliter of packed red blood cells was obtained from EDTA

    or anticoagulant-treated blood. The red blood cells were washed

    twice with 0.9% NaCI and lysed by addition of 1 ml of distilled

    water. The red cell membrane lysate was extracted with I ml CCI4,

    and the suspension was vortexed vigorously and then centrifuged for

    10 mm at 600 g. The aqueous supernatant was stored at -70#{176}C. Saliva

    for all experiments unless otherwise stated. Dialysis of the reaction

    mixture against acetate buffer did not increase significantly the

    removal ofsialic acid and therefore such dialysis was not used as the

    standard method for neuraminidase treatement of R protein.

    Consistent and reproducible results of isoelectric banding patterns

    following neuraminidase treatment were obtained when R proteins

    were labeled with “Co vitamin B12 before the enzyme treatment. The

    banding patterns of R proteins labeled after desialation by neur-

    aminidase were not as apparent as R proteins labeled before

    treatment with neuraminidase. The loss of affinity for the radiola-

    beled ligands following the treatment of neuraminidase was much

    greater in samples of saliva and granulocytes as compared with

    erythrocytes, amniotic fluid, and tears. Both acidic and basic compo-

    nents of desialated R proteins, however, appear to have almost the

    same degree of affinity for the vitamin B,2 ligands as the native

    forms of R proteins. The evidence for B12 affinity of desialated R

    isoproteins can be obtained from the observation that fresh saliva

    contains a significant amount of native desialated isoproteins (Fig.

    0 � a:

    -�-� 3

    to � u_ �



    -� -� �c� r�) �-,�5 Li�

    � c� � 0 w


    Human saliva was collected from healthy individuals. The sam-

    pIes were placed on ice within 30 mm of collection. The saliva was

    then centrifuged at 600 g for 20 mm at 4#{176}Cand the supernatant was

    stored at 70#{176}C.

    Amniotic Fluid (AF)

    Human amniotic fluid samples were o