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.
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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.
MATERIALS AND METHODS
.09
.9’
748 YANG. COLEMAN. AND DUPONT
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.
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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
