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

The Biochemical and Genetic Basis for the Microheterogeneity ofHuman 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 differentdegrees 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.

0006-4971/82/5904--0009$1.00/0

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.

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.

0�a:

-�-�3

to�u_�

C)

�:

-�-��c�r�)�-,�5 Li�

� c� �0w

I-

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 obtained from diagnostic

amniocenteses.

isolation of Granulocytes

Granulocytes were isolated as described by Willoughby et al.2’

Greater than 95% of the leukocytes were classified as granulocytes.

Their viability was consistently greater than 98% as judged by the

dye exclusion of trypan blue. Less than 0.2% residual erythrocytes

were found in the granulocyte suspension. The isolated granulocytes

were suspended in Hanks’ solution containing 0.1% CaCI2. The

concentration ofgranulocytes was then adjusted to 20 x l06/ml.

Release of R Proteins From isolated Granulocytes

The release of R proteins from isolated granulocytes was induced

by lithium treatment as described by Scott et al.’4 Briefly, LiCI

(0.2 M) was added to the isolated granulocyte suspension (20 x

106/ml) to yield a final concentration of 0.01 M of LiCI. The

mixture was left at room temperature overnight, and the supernatant

was collected after centrifugation and stored at - 70#{176}C.

Removal ofSialic Acids From R Proteins

Samples of R proteins were mixed with an equal volume of acetate

buffer (0.05 M sodium acetate, pH 5.5, containing 0.15 M NaCI,

0.1% CaCI2) and neuraminidase (500 U/mI, Behring Diagnostics,

Somerville, N.J.). The mixture was incubated at 37#{176}Cfor 16-24 hr

I 2 3 4 5 6 7.3

Fig. 1 . Thin-layer isoelectric focusing patterns of native Rtype vitamin B,2 binding proteins in human cells from blood andbody fluids. IEF was performed in 5% acrylamide gels containing5% glycerol. 1 .5% taurin (w/v). and 2% (w/v) ‘�Servalytes” over apH range of 2-8. Ten �diters of sample. labeled with 57Co-vitaminB,3, was applied to Whatman no. 1 . filter paper cut into 5 x 1 0 mmpieces. Electrofocusing was carried out for 3 hr at 1 O’C. The gelwas dried, then exposed to x-ray film at - 70’C for 2 wk. The pHgradient was determined by cutting the dried gel into strips (0.5 x10 cm). Each strip was eluted by soaking for 24 hr at roomtemperature in 1 .0 ml distilled water. and the pH values were thenmeasured directly. Sample identification: (1 ) Erythrocyte lysate(RBC); (2) amniotic fluid (16 wk of gestation) [AF (16 wk)J; (3)plasma from CML patient (PI (CML)]; (4) fresh saliva (Sal); (5)granulocyte R protein obtained by lithium ion stimulation ofisolated normal granulocytes (Gr); (6) amniotic fluid (39 wk ofgestation) IAF (39 wk)]; (7) tears.

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

A-TYPE VITAMIN B12 BINDING PROTEINS 749

I ). The banding patterns of the isoproteins with p1 > 4.5 occurring in

fresh saliva can be identified as clearly as the sialated isoproteins (p1

< 4.5). Similar observations were made for the affinity of R proteins

towards radioligands. The R proteins, once labeled with “Co vitamin

B2, did not lose the ligands for long periods of time at 4#{176}C.The

radiolabeled R proteins isolated via DEAE or Sephadex G-l 50

column chromatography could be identified on the IEF gel after

storage for 3 mo at 4#{176}C,although basic shifts of bands were observed

due to the spontaneous release of sialic acid from the more acidic R

proteins (data not shown). R proteins from saliva and granulocytes,

however, do lose their affinity for radioligands relatively quickly.

The IEF bands could not be detected when R proteins from

granulocytes and saliva were labeled with ‘7Co-B12 after incubation

at 37#{176}Cfor I 6 hr or after storage at 4#{176}Cfor 2-3 wk. R proteins from

erythrocytes were the most stable of all R proteins tested with

respect to their affinity for “Co-vitamin B,2.

Labeling Vitamin B,2 Binding Proteins

‘7Co-cyanocobalamin (specific activity 100-300 zCi/�g, 0.454

�ig/ml, Amersham, Arlington, Ill.) was diluted in phosphate-

buffered saline (pH 7.2) to final concentration of4.54 x 10’ pg/mI.

Samples of R proteins were mixed with an equal volume of “Co-B12

solution and incubated at 37#{176}Cfor I 5 mm. This radiolabeled

mixture was used for IEF gel electrophoresis.

Analytical Thin-Layer Isoelectric Focusing Gel (IEF)

Elect rophoresis

Analytical thin-layer isoelectric focusing in polyacrylamide gels

was accomplished by modification of the method described by

Karlsson et al.24 Polyacrylamide gels were made from an acrylamide

stock solution containing 29.1% (w/v) acrylamide and 0.9% (w/v)

N,N’-methylenebisacrylamide (Bio Rad Laboratories, Rockville

Center, N.Y.). The gel contained a final concentration of 5%

acrylamide, 5% glycerol, I .5% taurin (w/v, Eastman Co., Rochester,

N.Y.) and 2% (w/v) SERVALYTE (Accurate Chemical Scientific

Corp., Hicksville, N.Y.) yielding a pH range of 2-8 over the length

of the gel.

Samples ( 10 �l) (body fluids, cell lysates, etc.) were applied to

filter paper (Whatman no. I, England) strips 0.5 x I cm, aligned

with the electric field, and placed close to the cathode. Electrofocus-

ing was performed on an LKB 21 17 Multiphor apparatus for 3 hr at

5#{176}C.The voltage was slowly increased from 200 V to 1000 V during

the 3-hr electrofocusing. At the beginning of the electrofocusing, the

current was approximately 25mA and decreased during the run to 10

mA. Following electrophoresis, the gel was vacuum dried and

exposed to x-ray film (Kodak, Eastman, Rochester, N.Y. or Cronex,

Dupont Co., Minneapolis, Minn.) at - 70#{176}Cfor at least 7 days.

The p1-I gradient across the gel was determined by slicing the

dried gel (0.5 x 10cm), and eluting each slice with double distilled

water ( I ml) for 24 hr. and the pH values of the aqueous eluants were

measured directly.

DEAE Cellulose Chromatographyfor Separating TC

I and TC III

Chromatography was performed as described by Burger et al.2#{176}at

4#{176}Con a column ( I x 20 cm) packed with DEAE cellulose (DE-52),

Whatman, England). The column was equilibrated with 0.02 M

potassium phosphate, pH 7.5. The column was eluted with a linear

phosphate gradient in which the mixing solution contained 50 ml of

equilibrating buffer and the reservoir contained 50 ml of 0.3 M

KH2PO4, pH 4.5.

Preparation of Antiserum

Antiserum to human saliva was prepared by inoculating a rabbit

with a mixture containing equal volumes of fresh single donor saliva

(which had been previously centrifuged and filtered on 0.2 �m pore

filters; Gelman, Ann Arbor, Mich.) together with complete Freund’s

adjuvant. Rabbits received a total of 4 intracutaneous injections of

adjuvant mixture (I ml) at 10-day intervals, and the antibody was

tested using immunodiffusion plates against human saliva.

Immunodtffusion

Immunodiffusion tests were performed for “CO-B1 2-saturated

vitamin B2 binding proteins against the antiserum to human saliva

on 2% agarose gels in 0.05 M Veronal buffer, pH 8.6. After 24 hr.

excess free CO�B12 within the diffusion gel was removed by washing

with normal saline for 2 days, and the gel was then dried. The

radioactive precipitin lines were demonstrated by autoradiography.

Population and Family Studies

Samples (granulocytes, erythrocyte lysates, or saliva) from unre-

lated individuals in different populations were used in this study.

Paternity in the families studied was corroborated in all cases by

HLA typing and/or by red cell antigen typing.

Statistical Analysis

The genetic basis for the polymorphic variants of R protein was

tested using the Hardy-Weinberg theorem. The validity of the

Hardy-Weinberg equation for the proposed hypothesis was tested

using the chi-square (x2) test. The allelic segregation of R protein

variants was studied in randomly selected families, and the analysis

was applied to the distribution ofgenotypes among the siblings.

Results

JEF Gel Electrophoresis ofNative Vitamin B12

Binding Proteins

Figure 1 shows the autoradiogram of isoelectric

focusing (IEF) banding patterns of R proteins from

different tissue sources. The R proteins of granulocytes

and erythrocytes demonstrate very similar banding

positions within the pH range of 2.5-4. Plasma sample

of a patient with chronic myelogenous leukemia

(CML) gave a very strong band at pH 2.3. There were

no neuraminidase-resistant B,2 binding proteins

observed on IEF gels in the plasma samples obtained

from CML patients and normal individuals. The B,2-

unsaturated TC II, which is present in a considerable

amount in the plasma, could be demonstrated in the

same samples by autoradiogram following 7%-9%

acrylamide gel electrophoresis as described by Yang et

al.’8 (data not shown). The R proteins from saliva

revealed considerably more isoprotein bands across a

much wider pH range than R proteins from other

tissues and body fluids, and the isoelectric focusing

patterns of saliva R proteins varied considerably

depending on when sample collection occurred and the

quantity collected. For example, saliva samples

obtained after meals or late in the day or samples taken

in large quantities at one time (more than I ml) gave

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

a-U

FRACTION NUMBER

8010 20 30 40 50 60 70

Fig. 2. Elution profile of R proteins released from granulocytesof patients with chronic myelocytic leukemia (CML) on DEAEcellulose chromatography. Chromatography was performed usinga linear gradient system as described in Materials and Methods.Linear gradient elution was begun at tube 1 . R proteins released

from granulocytes from CML patients (“). TC Ill (--). and TC I

(---).

I 23456

Gr (CML) P1 (CML)

7 8 9 0

Gr (Normal)

Fig. 3. Autoradiogram of IEF gel for the R proteins fromnormal granulocytes and from both plasma and isolated granulo-cytes from patients with chronic myelocytic leukemia (CML). (1-3)R proteins released from granulocytes (Gr) from CML patients.(4-6) R proteins from plasma (P1) obtained from the same CML

patients as samples 1 -3. (7-1 1 ) R proteins from normal granulo-cytes (Gr).

750 YANG. COLEMAN, AND DUPONT

no isoprotein at pH 2.3. There were, however, bands of

increased intensity towards the higher pH range of the

gel with samples obtained under the above conditions.

Vitamin B,2 binding proteins in amniotic fluid (AF)

obtained at early pregnancy (prior to I 9 wk of gesta-

tion) were quite different from the R proteins

described above and displayed several isoprotein bands

in the pH range 5.16-5.75. In contrast, the vitamin B,,

binding proteins of AF obtained from pregnancy later

than I 9 wk of gestation yielded an unresolved pattern

that was very similar to the pattern obtained with

tears. The banding patterns of the native R proteins

were tissue specific (data not shown).

TC I and TC iii

Figure 2 shows the elution profiles of TC I and TC

III released in vitro from granulocytes from a patient

with CML using a linear gradient system on a DEAE

column. Two peaks of R proteins were observed (solid

line Fig. 2). One peak corresponded to TC III and the

other peak corresponded to TC I. The height of the TC

I peak is much greater in this sample than the peak of

TC III. The binding of endogenous vitamin B,2 to the

transcobalamins occurs extracellularly.”4 Since the

vitamin B,,-unsaturated TC I and TC III was obtained

in vitro from isolated granulocytes, the height of the

peaks may reflect the total amounts of TC I and TC

III. The TC I peak is not observed in the R proteins

released in vitro from normal granulocytes. The TC III

standard shown in Fig. 2 was obtained by normal

granulocyte stimulation in vitro with lithium chloride.

The TC I standard was isolated from the plasma of a

patient with CML by DEAE cellulose chromatogra-

phy using a discontinuous gradient system.7’25

The TC I and TC III protein obtained from granulo-

cytes of patients with CML can also be identified by

autoradiography of IEF gels. Figure 3 shows the

isoprotein patterns of R proteins from normal granulo-

cytes and the R proteins obtained from both plasma

and isolated granulocytes of patients with CML. The

R proteins from granulocytes of these patients con-

tamed both TC I (focused at pH 2.3) and TC III

(focused at pH 2.5-4), while the normal granulocytes

contain only TC III. Plasma from patients with CML

always contain vitamin B,2-unsaturated TC I and

sometimes TC III. Normal plasma contains a small

fraction of B,2-unsaturated TC I and trace amounts of

TC 111.2627 These studies indicate that the B,2-unsatu-

rated TC I occurring in plasma of CML patients

originates from the CML granulocytes, which have

increased sialation and secretion of R proteins.

Reaction ofR Proteins With Anti-human Saliva

Antibody

Antiserum against R proteins was prepared in rab-

bits (see Materials and Methods). This antiserum was

found to give only one precipitin line on immunodiffu-

sion plates analyzed by autoradiography after addition

of 57Co-vitamin B,,-labeled samples from saliva,

lysates from erythrocytes, granulocytes, tears, plasma

from patients with CML, or samples ofAF collected in

late pregnancy. No precipitin line was observed with

EDTA-treated normal plasma. It appears that autora-

diography of the immunodiffusion reactions may not

be sensitive enough to reveal the small amounts of

unsaturated TC I and TC III in EDTA-treated normal

plasma. The precipitin line of each of these different

samples showed reactions of identity with each other,

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

0�-.

(!� Q::c$)

cl)�

a,

0991

Saliva

Tears Gr

Anti-saliva Normal EDTA plasma

Fig. 4. Autoradiogram of an immunodiffusion plate. Eachsample was saturated with 57Co-CN B,2. Twenty-five zliters ofeach sample were introduced in the wells of a 2% agarose gelmade in 0.05 M veronal buffer. pH 8.9. The plate was kept in amoist chamber at room temperature for 24 hr of diffusion. Theexcess 57CO-CN B,2 in the diffusion plate was washed in 0.9%saline for 2 days and the gel was dried and exposed to x-ray filmfor 3 days. RBC: Erythrocyte lysate. Gr: Normal granulocyte.

R-TYPE VITAMIN B12 BINDING PROTEINS 751

indiciating that the R proteins in all samples were

immunologically indistinguishable (Fig. 4).

JEF Banding Patterns ofNeuraminidase-Treated R

Proteins

Neuraminidase treatment of R proteins obtained

from blood cells and body fluids revealed dramatic

changes in the patterns of bands observed by isoelectric

focusing and autoradiography. The banding patterns

of vitamin B,2 binding proteins following neuramini-

dase treatment were found to be identical in all sam-

pIes from the same individual regardless ofihe tissue

oforigin of the R proteins. Figure 5 demonstrates the

banding patterns of the R proteins following neur-

aminidase treatment of samples of blood cells, saliva,

and tears obtained from the same individual (the

plasma sample is, however, from an unrelated patient

with CML). Two major components of the R protein

with isoelectric point (p1) at 4.91 and 5.09 were

observed when the sialic acids had been removed. It

was noted that the component at p1 4.91 could be

further desialated to give rise to the form at p1 5.09.

This result could be obtained either by treatment of the

sample with higher concentrations of neuraminidase

and/or by means of longer incubation periods. These

more drastic incubation conditions, however, resulted

in the progressive disassociation of the radiolabeled

ligands from the R proteins, and the isoproteins of R

proteins therefore could not be detected. While the R

protein band with p1 5.09 probably is completely

desialated, the band with p1 4.91 may contain at least

PlasmaRBC (CML)

Fig. 5. Autoradiography of vitamin B12 binding proteins afterremoval of sialic acids by neuraminidase. Samples were labeledwith 57Co-vitamin B,, and then treated with neuraminidase at 37’Cfor 24 hr. Five percent polyacrylamide gels over a pH range 2-8were used. The dried gel was exposed to x-ray film for 14 days at

- 70’C. The R proteins were derived from: (1 ) Granulocytes (Gr);(2) erythrocytes (RBC); (3) saliva (Sal); (4) tears; (5) plasma (P1) of apatient with CML. All the samples were collected from the sameindividual, except the plasma of a patient with CML.

one neuraminidase-resistant sialic acid residue per

molecule. The relative resistance of the isoprotein (p1

4.91) to neuraminidase may be due to an R protein

conformation that limits the accessibility of neuramin-

idase to this remaining sialic acid residue.

Phenotypic Variations of R Protein Banding Patterns

in the Random Population

Isoelectric focusing of R proteins with samples from

different individuals after neuraminidase treatment

resulted in three different banding patterns. These

three R protein variants could be identified in samples

from granulocytes, erythrocytes, and saliva, and the

different R protein phenotypes are shown in Fig. 6.

Two distinct bands at p1 5.09 (the main desialated

protein) and 4.91 (the neuraminidase-resistant R pro-

tein) are seen in samples from certain individuals.

These two bands were designated according to the

nomenclature used for saliva R protein by Azen and

Denniston.22 The genetic locus coding for the R protein

was tentatively designated as TCR (transcobalamin R

protein). The most commonly observed R protein

phenotype in all populations tested was designated as

TCR I . Another set of bands slightly more basic than

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

5.09

4.91 11

I 234

Table 1 . R Protein Phenotypes in Different Tissues

of Single Individuals

NT. not tested.

Phenotypes 1 and 2 are the two allelic forms of the desialated A

proteins described in the text.

752 YANG. COLEMAN. AND DUPONT

Fig. 6. Phenotypes of R proteins from random donors ofgranulocytes (Gr), saliva (Sal). and erythrocytes (RBC). The threedifferent phenotypes are designated by the symbols 1 . 2. and 1 -2.57Co-vitamin B,2labeled samples were treated with neuraminidasefor 24 hr at 37’C before isoelectric focusing on 5% polyacrylamidegels containing 6% glycerol. 1 .5% taurin (w/v). and 2% “Serva-Iytes.�’ pH range 2-8. All phenotypes of the R proteins in salivasamples show complete acidic conversion (see text).

the two TCR I bands was designated as TCR 2. Of the

two isoprotein bands in TCR 2, the acidic band is not

as apparent as the alkaline band (see phenotypes

labeled 2 in Fig. 6). Similarly, the intensity of the

alkaline isoprotein band of the TCR 2 is not as strong

as that of the TCR I on IEF gels. Individuals possess-

ing a combination of the I and 2 were designated as

TCR 1-2. The isoprotein bands from saliva also show

an acidic shift that is probably attributable to glycosi-

dase enzymes present in saliva, but is not due to

neuraminidase activity. The acidic conversion of the

saliva R protein is shown in Fig. 7. All saliva samples in

Fig. 7 had been stored at -80#{176}C over 6 mo. Saliva

samples (nos. 2, 3, 4) treated with neuraminidase show

three different stages of R protein desialation, result-

ing in a situation in which sample no. 2 does not show

any acidic conversion, sample no. 3 is semiconverted,

and sample no. 4 shows complete conversion of the

desialated isoprotein, which is independent of neuram-

inidase activity. The acidic conversion of the isoprotein

at p1 5.09 can also be observed in saliva sample (no. 1)

without neuraminidase treatment. The three different

patterns of R protein bands that are observed in saliva

samples 2, 3, and 4 in Fig. 7 were obtained from saliva

samples with the same phenotype (TCR 1), and the

extent of the conversion cannot be predicted. The same

type of isoprotein conversion was also observed in R

proteins from erythrocytes and granulocytes, but only

when the 57Co-B, 2-labeled and neuraminidase-treated

samples were left at 4#{176}Cfor a prolonged period of time

(> 1-2 wk). As a result of these banding conversions in

saliva R proteins due to varying degrees of deglycosy-

lation, care should be taken when stored saliva samples

are used for phenotyping of R proteins. The banding

shift of R isoproteins is not always consistent in saliva

Fig. 7. Autoradiogram of saliva R protein on IEF gel. Samples2. 3. and 4 were treated with neuraminidase for 1 6 hr at 37’C whilesample 1 was untreated. These samples were obtained from 4different individuals of the same TCR phenotype. The samples hadbeen stored at -80’C for 6 mo before neuraminidase treatment.

samples, even when these are treated under standard-

ized conditions.

In order to examine whether the expression of R

protein phenotypes from different tissues within the

same individual is controlled by the same or by sepa-

rate genetic loci, the typing of R proteins from granu-

locytes, erythrocytes, and saliva from the same individ-

uals was carried out. The results are summarized in

Table 1 . Of 7 1 individuals tested, 5 1 were of the TCR

I, 18 of the TCR 1-2, and 2 of the TCR 2 type. Only

one R protein phenotype was observed for any individ-

ual regardless of which tissues or body fluids were

P��O; ofIndividuals

Tested

Phenotypes

SalivaGranulocytes Erythrocytes

4 1 1 1

4 1-2 1-2 1-2

2 2 2 2

39 1 1 NT9 1-2 1-2 NT

8 NT 1 1

5 NT 1-2 1-2

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

A-TYPE VITAMIN B12 BINDING PROTEINS 753

sampled, and all tissues for each individual displayed

one phenotype only, supporting the hypothesis that all

R proteins are controlled by only one genetic locus.

Population and Family Studies ofR Protein

Phenotypes

The simplest genetic model to explain the distribu-

tion of three R protein phenotypes in the population is

an autosomal mode of inheritance, where a single

genetic locus (called TCR) controls the expression of

two codominant alleles (TCR* I and TCR*2). Individ-

uals with only one pair of isoproteins (I or 2) are

assumed to be homozygous for the alleles at that locus,

and individuals with all four isoprotein bands (I -2) are

assumed to represent the heterozygous individuals.

Table 2 shows the observed and expected phenotype

frequencies and the gene frequencies obtained from a

random white population, an American black popula-

tion, and a Chinese population. Although there

appeared to be no significant differences in the pheno-

type frequencies found between males and females, the

gene frequencies differed significantly between dif-

ferent racial groups. The TCR*I was the most com-

mon allele in all populations tested. The TCR*2 was

observed at a frequency of I 2% in whites, but only at

I % among American blacks. Not a single individual in

the random Chinese population of 1 36 individuals

carried the TCR*2. Observed phenotype frequencies

in the white population did not differ significantly

from those expected by assuming Hardy-Weinberg

equilibrium (x2 = 1 .04; 0.5 < p < 0.75, d.f. = I).

One-hundred-one informative families with a total

of 329 children were studied for the segregation of the

electrophoretic variants of the TCR alleles I and 2.

The paternity of the children in each of these families

was corroborated by HLA typing. The family data are

summarized in Table 3. Unexpected variants different

from the I or 2 alleles were not observed in any of the

children in these families. The segregation ratio for the

different genotypes obtained in this family material

was consistent with a simple mendelian mode of inheri-

tance of two codominantly expressed alleles at a single

Table 3. Segregation of RP Alleles in White Families

Mating Matings Offspring 1 - 1 1 -2 2-2 x2 d.f.

1-1 x 1-2

1-2x1-2

1-2x2-2

1-lxl-1

1-1x2-2

33

3

1

62

2

103

6

2

215

3

55

2

-

215

-

48

3

2

-

3

-

1

0

-

-

0.476

-

-

-

-

1

-

-

-

-

Total 101 329 272 56 1 0.476 1

x2 0.476 . d.f. = 1, 0.50 < p < 0.75.

autosomal genetic locus (x2 = 0.476; 0.50 < p < 0.75,

d.f. = 1).

DISCUSSION

The banding patterns obtained for native 57Co-

B,2-labeled R proteins were studied by autoradiogra-

phy of IEF gel electrophoresis. Extensive microhetero-

geneity of R proteins from each tissue was found to be

due to differences in the amounts of sialic acid con-

tamed in the native R proteins. The banding patterns

were tissue specific and did not differ significantly

from individual to individual. Varying numbers of

narrowly spaced bands were found in the p1 range from

2.3 to 5.0. It is now generally believed that R proteins

from different sources are a mixture of isoproteins with

varying degrees of carbohydrate content, particularly

sialic acid.”3 However, the biochemical and genetic

relationship between TC I, TC III, and other R

proteins have not been documented. Stenman et al.28

postulated that serum TC I seen in chronic myeloid

leukemia came from granulocytes in which sialic acid

was added to TC III to make TC I. Stenman further

suggested that at least two different groups of R

proteins existed: one with acidic isoproteins (p1 2.3-

4.0) containing TC I and TC III occurring in all the

cells and fluids, and one more basic protein (p1 4.0-

5.0) only occurring in saliva and milk.5 Testing of this

hypothesis has brought conflicting results.21’29 Rachmi-

lewitz et al.2’ have suggested that as cells progress

through the myeloid maturation series, they shift from

the production of TC I to production of TC III, and

that TC I production is increased in the pathologic

states (e.g., CML) associated with proliferation of

T able 2. R Protein Pheno type Numbers and Gen e Frequencies in Random Populations

Population

Phenotypes Gene Frequencies

1 21 1 -2 2 Total

White

American black

Chinese

350(350.0)

47 (47.04)

136

98(95.5)

1 (0.95)

-

4(6.5)

0 (0.01)

-

452

48

136

0.88

0.99

1.00

0.12

0.01

0.00

White: x’ - 1.04. 0.5 < p < 0.75, d.f. 1.

Expected numbers (shown in parentheses) were calculated from the observed gene frequencies based on the Hardy-Weinberg equilibrium (p +

where p and q represent observed gene frequencies.

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

754 YANG. COLEMAN. AND DUPONT

immature myeloid cells. Hall,29 however, could not

find any evidence that granulocytes produce TC I.

Our study presents direct evidence that both TC I

and TC III originate from normal granulocytes and

granulocytes from CML patients. The present study

has also allowed the comparison of normal granulocyte

R proteins (TC III) released in vitro by lithium

stimulation with plasma TC I obtained from patients

with CML. These studies have also demonstrated that

all desialated R proteins from one individual have

identical IEF banding patterns, while the native R

proteins only differ with regard to sialic acid content.

The banding patterns of desialated R proteins, regard-

less of their tissue origins, were the same. The patterns

were very similar to those of desialated saliva R

proteins observed by Azen and Denniston.22 They

observed, however, an extra acidic band in samples

from leukocytes. We have not observed the extra acidic

57Co-B,2-labeled protein in samples obtained from lith-

ium-stimulated granulocytes.

The existence of the desialated R proteins in milk

and saliva may depend on two factors: the level of

sialyltransferase activity and the level of sialic acids in

a given tissue within which R protein is synthesized.

Another factor could be the number of galactose

residues that serve as acid acceptors for the terminal

sialic acid residues. Depending on the rate of salivation

versus the rate of glycosylation of R proteins, one

might consider that rapidly released glycoproteins may

be incomplete (lack of terminal sugar moieties) and

thus be undersialated. Such a hypothesis acquires

support from the observation by us (data not shown)

and other investigators3O3l that R proteins in milk and

saliva are much smaller in size (estimated by gel

filtration chromatography) compared with R proteins

from blood cells and other body fluids. Stenman5 has

also observed that the isoprotein population displaying

a high pI distribution (4.0-5.0) also possessed a

smaller average molecular size determined by gel

filtration. A marked difference in the carbohydrate

composition of R proteins from different tissue sources

has been reported, particularly with regard to the sialic

acid content.’9’2#{176} Plasma TC I, amniotic fluid, and

hepatoma cells containing R proteins had a relatively

high sialic acid content and a low content of fucose

residues, whereas saliva, milk, and TC III had a lower

sialic acid and higher fucose content. The carbohy-

drate composition of TC III and the normal granulo-

cyte vitamin B,2 binding protein have been reported to

be very similar, and these two proteins were found to

contain approximately half as much sialic acid and

twice as much fucose as TC I. This finding may

account for the fact that fucose and sialic acid are

interchangable as terminal sugar residues32 and that

the sialic acid residues sometimes are replaced by

fucose. Notwithstanding these facts, only sialic acid

moieties contribute to the net acidic charge of the

glycoprotein, and as a result, are entirely and uniquely

responsible for differential IEF gel patterns obtained

in this study.

The genetic study of polymorphism for the desial-

ated saliva R proteins by Azen and Denniston22 dem-

onstrated that the banding patterns obtained by IEF

gel electrophoresis could be explained by a simple

genetic model with one locus and two codominantly

expressed alleles. Our study also demonstrates that the

same genetic model applies for the desialated R pro-

teins from erythrocytes, granulocytes, and saliva. The

expression of the desialated TCR variants was always

the same when samples were taken from different

tissue sources of the same individual (Table 1 ). Fur-

thermore, the present study provides the biochemical

and immunochemical evidence that the same gene

controls the R protein production in different cells and

body fluids.

The polymorphic variants of R protein described in

this study have been observed in whites and American

blacks, but not in the Chinese population. The rare

TCR*2 occurs with a gene frequency of0.l2 in whites,

but only with a frequency of 0.0 1 in American blacks.

These findings are in agreement with the findings of

Azen and Denniston.22

Congenital deficiency of R proteins in two brothers

described by Carmel and Herbert33 can best be

explained by assuming that this disease is a simple

autosomal recessive or is X-linked. This particular

R-protein-deficient family was further studied by Hall

and Begley,34 who confirmed the total absence of R

proteins from any tissues (i.e., serum, saliva, gastric

juice, cerebrospinal fluid, and granulocytes) sampled

from these brothers. The present study provides the

biochemical and genetic documentation for the theory

that all desialated R proteins are genetically controlled

by a single locus.

ACKNOWLEDGMENT

Seth S. Horowitz is gratefully acknowledged for typing the

manuscript.

REFERENCES

I . Allen RH: Human vitamin B12 transport proteins. Prog Hema-

tol 9:57, 1975

2. Simons K, Gr#{228}sbeck R: Immunoelectrophoresis of human

gastricjuice. Clin Chim Acta 8:425, 1963

3. Jacob E, Baker SJ, Herbert V: Vitamin B12-binding proteins.

Physiol Rev6O:9l8, 1980

4. Stenman UH: Characterization of R-type vitamin B12-binding

proteins by isoelectric focusing. I. The relationship between TCI,

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

A-TYPE VITAMIN B12 BINDING PROTEINS 755

TCIII and the granulocytes R protein. Scand J Haematol 13:129,

I 974

5. Stenman UH: A characterisation of R-type vitamin B12-

binding proteins by isoelectric focusing. II. Comparisons of cobalo-

philin (R proteins) from different sources. Scand J Clin Lab Invest

35:147, 1975

6. Lawrence C: The heterogeneity of the high molecular weight

B2 binder in serum. Blood 33:899, 1969

7. Gizis EJ, Meyer LM: Separation of transcobalamins on small

DEAE-cellulose columns. Proc Soc Exp Biol Med 140:326, 1972

8. Bloomfield FJ, Scott JM: Identification of a new vitamin B12

binder (transcobalamin III) in normal human serum. Br J Haematol

22:33, 1972

9. Herbert V, Bloomfield FJ, Stebbins R, Scott J: Use of fluoride

to prevent erroneously high measurements of human serum unsatu-

rated B12-binding capacity (UBBC): Evidence that granulocyte-

derived binders (transcobalamin I and III) (TC I and TC III) are a

smaller component of normal circulating UBBC than previously

believed. J Clin Invest 52:39a, 1973 (abstr)

10. Hall CA: Transcobalamins I and II as natural transport

proteins ofvitamin B12. J Clin Invest 56:1 125, 1975

I I . Miller A, Sullivan iF: The in vitro binding of Co�#{176}labelled

vitamin B,2 in normal and leukemic serum. J Clin Invest 37:556,

I 958

I 2. England JM, Down MC, Wise Ii, Linnel JC: The transport of

endogeneous vitamin B12 in normal human serum. Clin Sci Mol Med

51:47, 1976

I 3. Gabuzda TG, Worm-Peterson J, Lous P: The binding of

vitamin B2 added in vitro and in vivo to normal human serum

proteins separated on ion exchange cellulose. Scand J Haematol

2:61, 1965

14. Scott JM, Bloomfield FJ, Stebbins R, Herbert V: Studies on

derivation of transcobalamin III from granulocytes: Enhancement

by lithium and elimination by fluoride of in vitro increments in

vitamin B,2-binding capacity. J Clin Invest 53:228, 1974

I 5. Bloomfield FJ, Weir DG, Scott JM: Some properties of

transcobalamin III from normal human serum. Br J Haematol

23:289, 1972

16. Daiger SP, Labowe ML, Parsons M, Wang L, Cavalli-Sforza

LL: Detection of genetic variation with radioactive ligands. III.

Genetic polymorphism oftranscobalamin II in human plasma. Am J

Hum Genet 30:202, 1978

I 7. Fr#{225}ter-Schr#{246}derM, Hitzig WH, Butler R: Studies on trans-

cobalamin (TC). I. Detection of TC II isoproteins in human serum.

Blood 53:193, 1979

I 8. Yang SY, Coleman P. Ochs H, Dupont B: Inheritance and

genetic linkage oftranscobalamin II. Hum Genet 57:307, 1981

19. Burger RL, Allen RH: Characterization of vitamin B12

binding proteins isolated from human milk and saliva by affinity

chromatography. J Biol Chem 249:7220, 1974

20. Burger RL, Mehlman CS, Allen RH: Human plasma R-type

vitamin B12-binding proteins. I. Isolation and characterization of

transcobalamin I, transcobalamin III, and the normal granulocyte

vitamin B12-binding protein. J Biol Chem 250:7700, 1975

21. Rachmilewitz B, Rachmilewitz M, Gross J: A vitamin B12

binder with transcobalamin I characteristics synthesized and

released by human granulocytes in vitro. Br J Haematol 26:557,

I974

22. Azen E, Denniston C: Genetic polymorphism of vitamin B12

binding (R) proteins of human saliva detected by isoelectric focus-

ing. Biochem Genet 17:909, 1979

23. Willoughby EW, Dupont B, Hansen JA, Good RA: The

indirect assay for leukocyte migration inhibitory factor (LIF)-

Standardization and the effect of pH. J Immunol Meth 22:99, 1978

24. Karlsson C, Davies H, Oman I, Anderson UH: LKB Applica-

tion note 75, 1975

25. Zittoun J, Zittoun R, Marquet J, Sultan C: The three

transcobalamins in myeloproliferative disorders and acute leukemia.

BrJ Haematol 31:287, 1975

26. Begley JA, Hall CA: Measurement of vitamin B12-binding

proteins of plasma. I. Technique. Blood 45:28 1 , I 975

27. Jacob E, Wong KJ, Herbert V: A simple method for the

separate measurement of transcobalamins I, II and III: Normal

ranges in serum and plasma in men and women. J Lab Clin Med

89:1 145, 1977

28. Stenman UH, Simons K, Gr#{228}sbeck R: Vitamin B,2 binding

proteins in normal and leukemic human leukocytes and sera. Scand J

Clin Lab Invest 21:202, 1968

29. Hall CA: The failure of granulocyte to produce transcobal-

amin I (TC I). Scand J Haematol 16:176, 1976

30. Gr#{228}sbeck R, Visuri K, Stenman UH: The vitamin B12 binding

protein in human saliva. Isolation, characterization and comparison

with unpurified protein. Biochim Biophys Acta 263:721, 1972

3 1 . Hippe E: Studies on the cobalamin-binding proteins of human

saliva. Scand J Clin Lab Invest 29:59, 1972

32. Dische Z: Fucose and sialic acid in glycoproteins of the

mucous of the digestive tract. Fed Proc 19:904, 1960

33. Carmel R, Herbert V: Deficiency of vitamin B12 binding

alpha globulin in two brothers. Blood 33:1 , 1969

34. Hall CA, Begley JA: Congenital deficiency of human R-type

binding proteinsofcobalamin. Am J Hum Genet 29:619, 1977

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

1982 59: 747-755  

SY Yang, PS Coleman and B Dupont type vitamin B12 binding proteinsThe biochemical and genetic basis for the microheterogeneity of human R- 

http://www.bloodjournal.org/content/59/4/747.full.htmlUpdated information and services can be found at:

Articles on similar topics can be found in the following Blood collections

http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requestsInformation about reproducing this article in parts or in its entirety may be found online at:

http://www.bloodjournal.org/site/misc/rights.xhtml#reprintsInformation about ordering reprints may be found online at:

http://www.bloodjournal.org/site/subscriptions/index.xhtmlInformation about subscriptions and ASH membership may be found online at:

  Copyright 2011 by The American Society of Hematology; all rights reserved.Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of

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