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The heterogeneity of human serum transferrin and human transferrin preparations on isoelectric focusing gels; no functional difference of the fractions in vitro
209
(‘CA 21 iS
The heterogeneity of human serum transferrin and human transferrin preparations on isoelectric focusing gels; no functional difference of the fractions in vitro
Summary The micro heterogeneity of the four main forms of human transferrin-Tf. TfFe(A), TfFe(B) and Tf.2Fe-as observed with the isoelectric focusing technique can be atrributed to the differences in sialic acid content. Transferrin molecules with the same iron content, but with varying sialic acid content--penta-sialo to asialo transferrins-show the same iron delivery to rat reticulocytes.
Introduction In previous papers [ 1--4j we described the separation and preparation of the four forms of human transferrin. Apotransferrin, monoferric transferrin A and B and diferric transferrin can be isolated by preparative isoelectric focusing. Using this technique with a narrow pH gradient of the ampholines, pH 5-7, we observed on analytical gels around the four main fractions, Tf. TfFe(A), TfFe(B) and Tf.2Fe. a number of bands which are in general weakly stained. On crossed immunoeIectrophoresis after isoelectric focusing of transferrin preparations, small peaks appeared of which the precipitation lines coincided with those of the main peaks. Human sera and human transferrin preparations fully saturated with iron also showed these multiple bands. The position of these minor diferric bands in the isoelectric focusing diagram interferes with those of the monoferric bands. As the intensity of the minor bands varies in the healthy and patients’ sera. e.g. alcoholics
210
[5], we investigated this micro heterogeneity and multiple band phenomenon more closely, in relation to the iron content, the sialic acid content and the removal of iron in vitro by reticulocytes. In this paper we present the separation, isolation. partial characterisation of these minor fractions. and the uptake of iron by cell suspensiona from the different diferric transferrins. Materials and methods All chemicals used were of analytical reagent grade. Human serum was obtained from healthy donors as well as from patients. Human transferrin prepared from serum from individuals and pooled serum was studied. Human transferrin was also obtained from Kabi (Sweden), lot No. 55926. Apotransferrin and iron transferrin were prepared as described earlier [ 11. Anti human transferrin was obtained from Dakopatts (Copenhagen. Denmarkj. Analytical and preparative isoelectric focusing were performed as described previously [ 1,2]. Neuraminidase Cl perfringens type V, lot No. 1036-8150-l with an activity of 0.2 U/mg protein using NAN-lactose is a Sigma product (USA). N-acetyl neuramic acid (sialic acid) cristalline type IV lot No. X2c’-1210 was obtained from Sigma (USA). Studies on the iron uptake by rat reticulocytes from diferric transferrins in vitro were performed as previously described in detail [3]. Trunsferrin from individual sera was obtained by affinity chromatography using a CNBr-Sepharose anti-human transferrin column [6]. Crossed immuno electrophoresis, following isoelectric focusing was carried out by the rapid procedure described by Skude and Jeppsson [7] at 20-25 V/cm. To control the stability of the diferric transferrin during desialylation. the desialylated fraction was separated from the neuraminidase by preparative isoelectric focusing and analysed from the E,,,/&,, ratio. and desialylated transferrins were The E470 and EzXu of diferric, monoferric measured in diluted solutions in order to obtain the E,,,,/Ezxo ratio. which is a characteristic of diferric and monoferric transferrins [9]. Amino acid analysis The amino acid composition of transferrins Tf(ZFe),. Tf(2Fe) + . Tf(ZFe),, and Tf(2Fe)_ was determined in quadruplicate as described earlier [8]. No significant differences were found. Desialylution The four diferric transferrins were completely desialylated with a neuraminidase solution with an activity of 6 U/ml. To 50 ~1 diferric transferrin, containing 3 mg Tf/ml 0.1 mol/l citrate, pH 6. 5 p.1 enzyme was added and incubated for 18 h at 37°C. To ensure that the transferrin was saturated during the analytical isoelectric focusing the pH was raised with 10 ~1 1 mol/l NaHCO,, prior to analysis.
211
Results To be able to isolate all the different diferric transferrin bands, the protein was theoretically over-saturated with iron up to 300%. Fig. 1 shows the isoelectric focusing pattern of partly saturated transferrin in the left column, Tf.2Fe, TfFe(B). TfFe(A) and apoTf are seen; in the second column the separation of the oversaturated preparation (200%) is seen. Just below the TfFe(B) position a Tf.2Fe band, which we call the Tf.(2Fe), band, is present; between this band and the main Tf.(2Fe), band a fraction called Tf.(2Fe)_ is observed. Below the main Tf.(2Fe), fraction another fraction is seen, transferrin four fractions of diferric called Tf.(2Fe)+ . From 400 mg over-saturated transferrin were isolated by preparative isoelectric focusing in a pH 5-7 gradient. The fractions obtained were purified by repeating the isoelectric focusing once more. In Fig. 1 these four separated fractions are seen, indicated as Tf.(2Fe)+ , Tf.(2Fe),, Tf.(2Fe)_ and Tf.(2Fe),. In Table1 the notation and the E,,,/E,,, is given for all these diferric transferrins and for TfFe(A) and TfFe(B); values are in agreement with literature data [9]. Fig.2 shows the crossed immuno electrophoresis of the over-saturated transferrin preparation. The numbers in Fig. 1 and Fig. 2 agree very well. The four diferric transferrins, Tf.2Fe+ , Tf.2Fe,,, Tf.ZFe- and Tf.2Fe, were made iron free and specifically labelled at the A or B site. In Fig. 3 the four bands of
Tf .
Tf.2Fe
:Tf We),
Tf Ok)+ 300% Fe +
o-
bA
Fig. I. Analytical isoelectric focusing from left to right. Tf 80% saturated. l-f 200% over-saturarcd Tf.(ZFe) + , Tf.(ZFe),, Tf.(2Fe)_. Tf.(ZFe),, TfFe(A) and Tf 300% over-aaturatcd. Direction of migration from top (-) to bottom (+). pH gradient top. pH 6.7. bottom. pH 5.2.
THE
RATIO
C,:,,
I:,,,,
(Tf 7FC).
0.043
(Tf.2FC),,
0 045
(TI” 21-c)
0 045
(1-f 7FC)
0 044
l-f. I Fe( 13)
0 025
Tf. I Fe(A)
O.O?.h
FOR
SEL’l:.RAL.
TRAI\;Sl~ERRIN
PRI;I’ARATIONS
Fig. 2 Crossed immunoelcctrophoresis after CocuGng of iron saturated Tf.(2Fet _ , Tf.(ZFe),,. Tf.(2Fe) and Tf.(ZFe),.
serum. the Icur dlfcnc
tr;lnsferrin~.
P
,c
0
-
2
b,,+
0
-
B
b,
(t
0
-
b,
A
Fig. 3. The four diferric transferrins (indicated by i. 0. - and b) and the H and A specific monofcrrk trensferrins. derived from the diferric fractions. The mnnofcrric transfwrins 31-t‘ contaminated with diferric transferrins.
diferric, monoferric B and monoferric A transferrins are shown. The differences in isoelectric point between the four fractions is found in all forms of transferriu (also present, but not shown, in apotransferrin). When .the four diferric transferrins are incubated with increasing activity of neuraminidase, they gradually lose their sialic acid and the differences in isoelectric focusing. Fig. 4 shows the four diferric transferrins, indicated by +. 0, - and b respectively in their original diferric state and after extensive desialylation, together with two partial desialylated fractions (0.1-h incubation). After extensive desialylation the differences in mobility disappeared, in agreement with observations in the literature [IO]. This finding suggests strongly that the differences in isoelectric focusing between the four fractions are caused by siahc acid content. Carbohydrate analyses are under study now. The four diferric fractions. Tf(:“Fe),. Tf(:‘Fe)_ . Tf(yFe),, and Tf( YFe) _ were
714
U,l o,?
-f-o-
6
6
6
011 18 18
b oo+o-b
6
6
18
18
U/ml h
Tf
incubated with rat reticulocytes. The iron uptake (‘YFe) was measured during a period of 60 rnin and presented in Fig. 5. It is shown that there exists no significant difference between the four human transferrins in delivering its iron to rat reticufocytes. Discussion and conclusions (1) Preparation of transferrin, obtained from human pooled or individual sera. show micro heterogeneity in isoelectric focusing. Besides the four main fractions. dependent on the degree of saturation (and pH of Iabelling) Tf. TfFe(A), TfFe(B) or Tf.2Fe. minor fractions around these main fractions can be observed. (2)These minor fractions around the major fraction Tf.2Fe were isolated. Based
I.
0
4
I
15
60
30 Incubation time (min
Fig. 5. Uptake of iron by rat reti~uI~~~tc~ Tf.(tFe) _ : Cl, Tf.(2Fe) i ; A, Tf.(2Fe),.
from
idated
)
Tf.yFr
ptrparatmns.
0,
Tf.(lFe),,:
A.
on the ratio E470/E2A0 at least four diferric fractions could be isolated. From these four diferric fractions the apo, monoferric A and B transferrins were prepared. The mutual distance between these fractions on the isoelectrical focusing gel was still the same. (3) The four diferric fractions called Tf.2Fe+ , Tf.2Fe0, Tf.2Fe. and Tf.2Fe, do not differ in amino acid composition, but do differ in sialic acid content on account of their similarity in isoelectric point after des~alyIation. They represent possibly di-sialo, tri-sialo, tetra-sialo and penta-siaio diferric transferrin. (4) Literature values for the carbohydrate chains of transferrins [ 1 l] suggest the existence of transferrin molecules with two bi-antennary and molecules with one bi-antennary and one tri-antennary branched hetero-saccharide chain, each with a sialic acid residue at the end. Thus, the presence of asialo, mono-. di-, tri-, tetra- and penta-sialo transferrins in serum is possible. The major diferric transferrin is a tetra-sialo transferrin. The
abnormal transferrin band in alcoholics observed by Stibler et al could be a di-sialo transferrin [5]. (5) The binding of the different diferric transferrins with rat reticulocytea and the intracellular iron uptake is equal. It seems likely that the carbohydrate chain is not directly involved in the binding with the specific membrane receptor [3]. References I Van EiJk HG. Van Noort WL, Kroob MJ. Van der Hcul C‘. Analy& of the Iron-bIndIng bltcz (11 tranafrrrin by isoelectric focusing. J Clin Chcm Clin Biochem lY7X: Ih: 557-560 7 Van EiJk HG. Van Noort WL. Kroos MJ. Van der Hwl C. Isolation of the two monofcrric human tranbferrins by preparative iaoclectric focusing. J Clin Chem Clin Riochem IYXO: IX: 563 566. 3 Van der Heul C, Krooa MJ. Van Noort WL, Van EiJk H<;. No functional dlffcrence of the I\+o Iron-binding sites of human tranaferrin In vitro. Clin Sci 19X1; 60: 1X5-190. 3 Van der Hcul C. Some aspcctc of the iron uptake by crythrold cells. Rotterdam: Ernstnu\ Uni\cr\a\l\. I YXI ; Thesis. 5 Stibler H. Sydow 0. Berg S. Quantitative satlmation of abnormal micro hctcrogenclt\ of xcrum transfcrrin in alcoholics. Pharmacol Biochem Behav 19X0: 13. Suppl I : 47-5 I 6 Van EiJk HG. Van Noort WL. Isolatwn of rat tranafcrrin wing CNBr activated Scpharnac-4B J (‘lln (‘hem Clin Biochern lY76: 14: 475-47X 7 Skudc Ci. Jrppaaon JO. Thin lager electrofocu.\ing followed hy elcctrophorcala In antihod\ contanlng gel. Sand J Chn Lab Invest iY72: 29. huppl 124: 55-54. X Verhocf NJ, Van EiJk HG. Isolation. charactrriratlon and functlvn of cord-hlcx)d trnn\fcrt-In. (lln %I Mel Med 1975; 4X: 335-340. Y Frxden E. Aisen Ph. Forms of Iron tramfcrrlns. Trend5 Blochcm Sci IYXO: 5 S1 IO Hamann A. Micro hetrrogcncity of sxxm~ glyoprotcins ;I?. raealcd h\ flat-hcd gel I\ocIcctrII focusing. In: Radola RJ, Gracaalin 13. cd> Elcctro focusing and iw tnchophowib Berlin-Ncv. York W. de <;ruytcr. IY77: ?ZY--335. I t Spik <;. New results concerning the structure. the conformation and the hiotoglcal rola of gtycam of different transfcrrins. In: Saltman P. Hcgrnauer J. eds. Proteins of Iron \torage and transport Amsterdam:
Elsevier.
in pres:.
(‘CA 21 iS
The heterogeneity of human serum transferrin and human transferrin preparations on isoelectric focusing gels; no functional difference of the fractions in vitro
Summary The micro heterogeneity of the four main forms of human transferrin-Tf. TfFe(A), TfFe(B) and Tf.2Fe-as observed with the isoelectric focusing technique can be atrributed to the differences in sialic acid content. Transferrin molecules with the same iron content, but with varying sialic acid content--penta-sialo to asialo transferrins-show the same iron delivery to rat reticulocytes.
Introduction In previous papers [ 1--4j we described the separation and preparation of the four forms of human transferrin. Apotransferrin, monoferric transferrin A and B and diferric transferrin can be isolated by preparative isoelectric focusing. Using this technique with a narrow pH gradient of the ampholines, pH 5-7, we observed on analytical gels around the four main fractions, Tf. TfFe(A), TfFe(B) and Tf.2Fe. a number of bands which are in general weakly stained. On crossed immunoeIectrophoresis after isoelectric focusing of transferrin preparations, small peaks appeared of which the precipitation lines coincided with those of the main peaks. Human sera and human transferrin preparations fully saturated with iron also showed these multiple bands. The position of these minor diferric bands in the isoelectric focusing diagram interferes with those of the monoferric bands. As the intensity of the minor bands varies in the healthy and patients’ sera. e.g. alcoholics
210
[5], we investigated this micro heterogeneity and multiple band phenomenon more closely, in relation to the iron content, the sialic acid content and the removal of iron in vitro by reticulocytes. In this paper we present the separation, isolation. partial characterisation of these minor fractions. and the uptake of iron by cell suspensiona from the different diferric transferrins. Materials and methods All chemicals used were of analytical reagent grade. Human serum was obtained from healthy donors as well as from patients. Human transferrin prepared from serum from individuals and pooled serum was studied. Human transferrin was also obtained from Kabi (Sweden), lot No. 55926. Apotransferrin and iron transferrin were prepared as described earlier [ 11. Anti human transferrin was obtained from Dakopatts (Copenhagen. Denmarkj. Analytical and preparative isoelectric focusing were performed as described previously [ 1,2]. Neuraminidase Cl perfringens type V, lot No. 1036-8150-l with an activity of 0.2 U/mg protein using NAN-lactose is a Sigma product (USA). N-acetyl neuramic acid (sialic acid) cristalline type IV lot No. X2c’-1210 was obtained from Sigma (USA). Studies on the iron uptake by rat reticulocytes from diferric transferrins in vitro were performed as previously described in detail [3]. Trunsferrin from individual sera was obtained by affinity chromatography using a CNBr-Sepharose anti-human transferrin column [6]. Crossed immuno electrophoresis, following isoelectric focusing was carried out by the rapid procedure described by Skude and Jeppsson [7] at 20-25 V/cm. To control the stability of the diferric transferrin during desialylation. the desialylated fraction was separated from the neuraminidase by preparative isoelectric focusing and analysed from the E,,,/&,, ratio. and desialylated transferrins were The E470 and EzXu of diferric, monoferric measured in diluted solutions in order to obtain the E,,,,/Ezxo ratio. which is a characteristic of diferric and monoferric transferrins [9]. Amino acid analysis The amino acid composition of transferrins Tf(ZFe),. Tf(2Fe) + . Tf(ZFe),, and Tf(2Fe)_ was determined in quadruplicate as described earlier [8]. No significant differences were found. Desialylution The four diferric transferrins were completely desialylated with a neuraminidase solution with an activity of 6 U/ml. To 50 ~1 diferric transferrin, containing 3 mg Tf/ml 0.1 mol/l citrate, pH 6. 5 p.1 enzyme was added and incubated for 18 h at 37°C. To ensure that the transferrin was saturated during the analytical isoelectric focusing the pH was raised with 10 ~1 1 mol/l NaHCO,, prior to analysis.
211
Results To be able to isolate all the different diferric transferrin bands, the protein was theoretically over-saturated with iron up to 300%. Fig. 1 shows the isoelectric focusing pattern of partly saturated transferrin in the left column, Tf.2Fe, TfFe(B). TfFe(A) and apoTf are seen; in the second column the separation of the oversaturated preparation (200%) is seen. Just below the TfFe(B) position a Tf.2Fe band, which we call the Tf.(2Fe), band, is present; between this band and the main Tf.(2Fe), band a fraction called Tf.(2Fe)_ is observed. Below the main Tf.(2Fe), fraction another fraction is seen, transferrin four fractions of diferric called Tf.(2Fe)+ . From 400 mg over-saturated transferrin were isolated by preparative isoelectric focusing in a pH 5-7 gradient. The fractions obtained were purified by repeating the isoelectric focusing once more. In Fig. 1 these four separated fractions are seen, indicated as Tf.(2Fe)+ , Tf.(2Fe),, Tf.(2Fe)_ and Tf.(2Fe),. In Table1 the notation and the E,,,/E,,, is given for all these diferric transferrins and for TfFe(A) and TfFe(B); values are in agreement with literature data [9]. Fig.2 shows the crossed immuno electrophoresis of the over-saturated transferrin preparation. The numbers in Fig. 1 and Fig. 2 agree very well. The four diferric transferrins, Tf.2Fe+ , Tf.2Fe,,, Tf.ZFe- and Tf.2Fe, were made iron free and specifically labelled at the A or B site. In Fig. 3 the four bands of
Tf .
Tf.2Fe
:Tf We),
Tf Ok)+ 300% Fe +
o-
bA
Fig. I. Analytical isoelectric focusing from left to right. Tf 80% saturated. l-f 200% over-saturarcd Tf.(ZFe) + , Tf.(ZFe),, Tf.(2Fe)_. Tf.(ZFe),, TfFe(A) and Tf 300% over-aaturatcd. Direction of migration from top (-) to bottom (+). pH gradient top. pH 6.7. bottom. pH 5.2.
THE
RATIO
C,:,,
I:,,,,
(Tf 7FC).
0.043
(Tf.2FC),,
0 045
(TI” 21-c)
0 045
(1-f 7FC)
0 044
l-f. I Fe( 13)
0 025
Tf. I Fe(A)
O.O?.h
FOR
SEL’l:.RAL.
TRAI\;Sl~ERRIN
PRI;I’ARATIONS
Fig. 2 Crossed immunoelcctrophoresis after CocuGng of iron saturated Tf.(2Fet _ , Tf.(ZFe),,. Tf.(2Fe) and Tf.(ZFe),.
serum. the Icur dlfcnc
tr;lnsferrin~.
P
,c
0
-
2
b,,+
0
-
B
b,
(t
0
-
b,
A
Fig. 3. The four diferric transferrins (indicated by i. 0. - and b) and the H and A specific monofcrrk trensferrins. derived from the diferric fractions. The mnnofcrric transfwrins 31-t‘ contaminated with diferric transferrins.
diferric, monoferric B and monoferric A transferrins are shown. The differences in isoelectric point between the four fractions is found in all forms of transferriu (also present, but not shown, in apotransferrin). When .the four diferric transferrins are incubated with increasing activity of neuraminidase, they gradually lose their sialic acid and the differences in isoelectric focusing. Fig. 4 shows the four diferric transferrins, indicated by +. 0, - and b respectively in their original diferric state and after extensive desialylation, together with two partial desialylated fractions (0.1-h incubation). After extensive desialylation the differences in mobility disappeared, in agreement with observations in the literature [IO]. This finding suggests strongly that the differences in isoelectric focusing between the four fractions are caused by siahc acid content. Carbohydrate analyses are under study now. The four diferric fractions. Tf(:“Fe),. Tf(:‘Fe)_ . Tf(yFe),, and Tf( YFe) _ were
714
U,l o,?
-f-o-
6
6
6
011 18 18
b oo+o-b
6
6
18
18
U/ml h
Tf
incubated with rat reticulocytes. The iron uptake (‘YFe) was measured during a period of 60 rnin and presented in Fig. 5. It is shown that there exists no significant difference between the four human transferrins in delivering its iron to rat reticufocytes. Discussion and conclusions (1) Preparation of transferrin, obtained from human pooled or individual sera. show micro heterogeneity in isoelectric focusing. Besides the four main fractions. dependent on the degree of saturation (and pH of Iabelling) Tf. TfFe(A), TfFe(B) or Tf.2Fe. minor fractions around these main fractions can be observed. (2)These minor fractions around the major fraction Tf.2Fe were isolated. Based
I.
0
4
I
15
60
30 Incubation time (min
Fig. 5. Uptake of iron by rat reti~uI~~~tc~ Tf.(tFe) _ : Cl, Tf.(2Fe) i ; A, Tf.(2Fe),.
from
idated
)
Tf.yFr
ptrparatmns.
0,
Tf.(lFe),,:
A.
on the ratio E470/E2A0 at least four diferric fractions could be isolated. From these four diferric fractions the apo, monoferric A and B transferrins were prepared. The mutual distance between these fractions on the isoelectrical focusing gel was still the same. (3) The four diferric fractions called Tf.2Fe+ , Tf.2Fe0, Tf.2Fe. and Tf.2Fe, do not differ in amino acid composition, but do differ in sialic acid content on account of their similarity in isoelectric point after des~alyIation. They represent possibly di-sialo, tri-sialo, tetra-sialo and penta-siaio diferric transferrin. (4) Literature values for the carbohydrate chains of transferrins [ 1 l] suggest the existence of transferrin molecules with two bi-antennary and molecules with one bi-antennary and one tri-antennary branched hetero-saccharide chain, each with a sialic acid residue at the end. Thus, the presence of asialo, mono-. di-, tri-, tetra- and penta-sialo transferrins in serum is possible. The major diferric transferrin is a tetra-sialo transferrin. The
abnormal transferrin band in alcoholics observed by Stibler et al could be a di-sialo transferrin [5]. (5) The binding of the different diferric transferrins with rat reticulocytea and the intracellular iron uptake is equal. It seems likely that the carbohydrate chain is not directly involved in the binding with the specific membrane receptor [3]. References I Van EiJk HG. Van Noort WL, Kroob MJ. Van der Hcul C‘. Analy& of the Iron-bIndIng bltcz (11 tranafrrrin by isoelectric focusing. J Clin Chcm Clin Biochem lY7X: Ih: 557-560 7 Van EiJk HG. Van Noort WL. Kroos MJ. Van der Hwl C. Isolation of the two monofcrric human tranbferrins by preparative iaoclectric focusing. J Clin Chem Clin Riochem IYXO: IX: 563 566. 3 Van der Heul C, Krooa MJ. Van Noort WL, Van EiJk H<;. No functional dlffcrence of the I\+o Iron-binding sites of human tranaferrin In vitro. Clin Sci 19X1; 60: 1X5-190. 3 Van der Hcul C. Some aspcctc of the iron uptake by crythrold cells. Rotterdam: Ernstnu\ Uni\cr\a\l\. I YXI ; Thesis. 5 Stibler H. Sydow 0. Berg S. Quantitative satlmation of abnormal micro hctcrogenclt\ of xcrum transfcrrin in alcoholics. Pharmacol Biochem Behav 19X0: 13. Suppl I : 47-5 I 6 Van EiJk HG. Van Noort WL. Isolatwn of rat tranafcrrin wing CNBr activated Scpharnac-4B J (‘lln (‘hem Clin Biochern lY76: 14: 475-47X 7 Skudc Ci. Jrppaaon JO. Thin lager electrofocu.\ing followed hy elcctrophorcala In antihod\ contanlng gel. Sand J Chn Lab Invest iY72: 29. huppl 124: 55-54. X Verhocf NJ, Van EiJk HG. Isolation. charactrriratlon and functlvn of cord-hlcx)d trnn\fcrt-In. (lln %I Mel Med 1975; 4X: 335-340. Y Frxden E. Aisen Ph. Forms of Iron tramfcrrlns. Trend5 Blochcm Sci IYXO: 5 S1 IO Hamann A. Micro hetrrogcncity of sxxm~ glyoprotcins ;I?. raealcd h\ flat-hcd gel I\ocIcctrII focusing. In: Radola RJ, Gracaalin 13. cd> Elcctro focusing and iw tnchophowib Berlin-Ncv. York W. de <;ruytcr. IY77: ?ZY--335. I t Spik <;. New results concerning the structure. the conformation and the hiotoglcal rola of gtycam of different transfcrrins. In: Saltman P. Hcgrnauer J. eds. Proteins of Iron \torage and transport Amsterdam:
Elsevier.
in pres:.