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Preparation of a glycopeptide from an immunoglobulin kappa polypeptide chain from the mouse
3 9 (}
BIOCHI.~,tlCA I';T B I O P H Y S I C . \
PRELIMINARY
A{;TA
NOTES
BBA 3 I O I 7
Preparation of a glycopeptide from an immunoglobulin kappa polypeptide chain from the mouse We recently reported that a plasma cell t u m o u r (MOPC 4 6) which was induced in I~IALB/e micO -s secretes a kat)pa-type light chain with a relatively high cart)ohydrate content *. This is an unusual finding since light chains are generally associated with only small amtmnts of carbohydrate. In the present paper we describe further observations on this protein. In the first place we have analysed the sugar contents of proteins obtained from tumours at two different transfer generations (G 4 and Ga.0 and the results indicated that they were essentially the same (Table I). These data {lifter from those which were ret)orted t)y MELCHERS, I.F,CNNOXAND I?A('{)N 9 for a similar protein produced by an{}ther transplant line of the same tumour. We have also isolated :t glycopeptide from the protein. TAI~LE
I
CARBOHYDR.~.TI';
{:ON'rENT
TXVO G E N E R A T I O N S
T u r n o u t generalio~
134 {;a:,
OF
KAI'PA-TYPE
(}F TUMOUR
LIGIIT
{ tIAIN
PRO1)UtTEI)
BY MOUSE
TIJMOUR
.M()PC 4 6
A'F
TRANSPLANT
Carbohydrate content (g reh'ased per soo g prote~n) Hexose *
th, aosamine""
S i a l i c acid" * *
-t-5.t -t.3-'
2.88 2.74
o.9I o.85
* Mannose and galactose. The values were obtained by an orcinol-sulphuric a n d i n c l u d e a c o n t r i b u t i o n d u e t o t h e s m a l l a m o u n t of f u c o s e ~ (o.34 % ) p r e s e n t . * * Estimated as glucosamineL * * * M e a s u r e d a s N - a c e t y l n e u r a m i n i c a c i d ~.
acid method s
All the carbohydrate moved with the protein when tile latter was subjected to electrophoresis (pit 8.0) on cellulose acetate "). Moreover, if the protein were pretreated with 8 M urea, two protein bands of apt}roximately equal intensity appeared after electrt}phoresis, both of which stained with periodate-Schitt reagents alsolL These experiments SUpl)ort our belief that the carbohydrate which we have found occurs as an integral part of the light chain. The follmving procedure was used in order to isolate a glycopeptide from the protein (transplant generation 35). The glycoprotein (5o rag) was dissolved in 5 ml o.o5 M t)hosphate buffi:r, pH 7.5, which contained o.oi M EI)TA. bIercurit)apain (t rag; Sigma Laboratories, Lot 55 B;'I5OO) was added and the solution was maintained at 5 o~ and ptt 7.5. By following the course of the reaction with a t)ht}tometric nmhydrin method lz it was shown that r e a c t i o n was complete after about 2 h when a b o u t Hiochim. t¢iophys..-Iota, ]47 (x{) ~71 39{' 398
PREI.IMINARY
397
NOTES
25 O,.oof the peptide bonds of the protein had been cleaved. The pH of the digest was adjusted to 5.5:1.63 mg bound hexose were present at this stage. Ethanol (Io ml) was added to the solution and the small amount of precipitate which formed was removed by eentrifugation, leaving ~.32 nag hexose in the supernatant. The latter was taken to dryness i n vacuo and the residue extracted with successiw." i5-ml fractions of ethanol containing increasing amounts of water. The fraction e.xtracted with water-.ethanol (2o:8o, v/v) contained o.4 nag hexose and, judging by photometric ninhydrin assays, was least contaminated with peptides which did not contain carbohydrate. It contained about 2o moles of peptide lacking carbohwtrate for each mole of glyc opeptide. This fraction was further purified by chromatography on Dowex 5o-X8 (H~), with water as eluting agent Is,'4. About (}o % of the carbohydrate applied to the column was recovered. This glycopeptide was analysed by column chromatography after acid hydrolysis for its amino acid and amino sugar contents (Table II). It was also shown to "fA BIA'; II AMINO
ACID
LIGHT
CHAIN
AND
AMINO
PRODUCED
SUGAR
ANALYSIS
BY MOUSE
Component
Asl>artic acid * Serine Glutamic acid Glycine Isoleucine Glucosamine
OF
"FUblOUR
A (H.YCOPEPTIDE
ISOLATED
FROM
THE
KAPPA-TYPE
MOPC 40
Hydrolysis conditions 6 3I tlCI; 1zoO; I6 h
4 31 IIC); IOO°; 3 h
x .o o.97 o.88 o.38 o.38 2.o 3
i .o o.87 I. x 7 o.52 o.39 3.~
* Taken as standard.
contain 4.2 moles hexose per mole of aspartic acid. The glycopeptide gave rise to mannose and galactose on acid hydrolysis (2 M HC1; IOO~; 3 h), the former in slightly larger amounts. Small quantities of fucose were also shown to be present by paper chromatography (ethyl acetate-pyridine-water (12:5:4, by vol.)). The amino sugar was identified as glucosamine by the column chromatographic techniques employed. The nature of the carbohydrate-pel)tide bond in this protein has not been rigorously established yet but it is likely to be 2-acetamido-I-(fl-I.-aspartanfido)-i,2dideoxy-fl-D-glucose as in certain other glycoproteins Is. Thus treatment of the whole protein with o.~ M NaOH for 24 h at room temperature did not result in a splitting of the carbohydrate from the polypeptide chain. 2\ linkage involving the hydroxyl grou t) of serine is thus excluded 'n. Of the other amino acids identified in the glycopeptide only aspartic acid, or more correctly asparagine, is likely to provide the point of attachlnent of the carbohydrate moiety to the polypeptide chain, by analogy with other glycotlroteins. Moreover the only sugar yet shown to be involved in a linkage to asparagine in a glycoprotein is N-acetyl-l)-glucosamine'~. It is relevant to mention that a similar protein produced by tumour MOPC 41 has the sequence -Ser-Gln-Asx-Ile-Gly-- at positions 26-3o inclusiw~ froln the Ihoch¢m. t3iophys. Acta, Lt7 ( i 9 0 7 ) 3 9 6 - 3 9 8
398
I>RI.;I.I MI NAIC~" N() I'E5
N - t e r m i n a l e n d of t h e p o l y p e p t i d e c h a i n ' L It is ilot u n r e a s o n a b l e to s u g g e s t , f r o m a c o n s i d e r a t i o n of t h e a m i n o acid a n a l y s i s of o u r glycotw.ptide ( ' f a b l e II) t h a t 1he c a r b o h v d r a t c I n o i e t v w a s origin;ally a t t a c h e d a t a n asparagilu', r e s i d u e w h i c h ~)t'Cllrs in a s i m i l a r p - s i t | o n in t h e g l y c o p r o t r i n . T h e n o n - i n t c , ~ r a l \;titles f o u n d for l)t~tlt g l y c i l w a n d i s o l e u c i n c m o s t p r o b a b l y a r o s e b e c a u s e of p a r t i a l t'lc~tvage, b v t h e p a t ) a i n , ,)f t h e l i n k a g e b e t w e e n lie a n d t h e ASh r e s i d u e c a r r y i n g t h e c a r b o h y d r a t e m o i r t v . P a r t i a l c n z v m a t i c cle;tvage of l)t,ptide b o n d s in a n a l o g o u s p o s i t i o n s in o t h e r g l y c o p r o t c i n s
h~ts SOlllt'tinlcs ])e(!n ()])SCl\'t'd Is. It was m e n t i o n e d a b o v e t h ; t t a s i m i l a r p r o t e i n p r t ) d u c e d b y a n o t h e r t r a n s p l a n t line of the. s a m e t u r n o u t w a s also a glycoprotein but that it c o n t a i n e d m o r e carl)oh y d r a t e t h a n is p r e s e n t in . u r preparations..'~IEI.CHERS, LI-NNOX .\NI) FACO.X~ f o u n d t h a t t h e i r g l y c o p r o t c i n c t m l d be s p l i t i n t o t h r e e c o m p o n e n t s b y c h r o m a t o g r a p h y on I ) l ' . - \ E - e e l h f l o s e . T h e s e c o m p o n e n t s d i f f e r e d in t h e i r sialic acid c o n t e n t s w h i c h were s h o w n to b e a p l ) r o x , o, I, a n d 2 rcsi(lues rest)ectively'. F.ach (,f t h e s e c h r o m a t o g r a p h i c f r a c t i o n s w a s r e p o r t e d t o c o n t a i n a b o u t 4 r e s i d u e s e a c h (,f nl~tnll()S(! a n d of g a l a c t , s c , 2 of fucose an(l {) of h e x o s a m i m ' , a m o u n t s w h i c h a r e a b o u t t w i c e tll,~sc, w h i c h we lind in o u r proteins ( T a b l e I). I t s h o u l d also b e m e n t i o n e d t h a t t h e t r y p t o l ) h a n c o n t e n t w h i c h we h a v e f o u n d (3.5 residues) ~:' is l o w e r t h a n t h a t r e p o r t e d b y .MELCHH{S, LE.x'.x:C X ..X.','l) I:..\CoX" in t h e i r t h r e e f r a c t i o n s (5.4 to 5.6 residues). T h e r e a s o n s for t h e s e d i f f e r e n c e s a r e n o t v e t c]car. V e r y r e c e n t l y M1..I.cIH.;RS AND I(xt)l'l :e° h a v e r e p o r t e d t h e p r e p a r a t i o n . f a g l y c o p c p t i d e f r o m a t r y p t i c d i g e s t of t h e i r l i g h t c h a i n .
Department of ('heroical l'athology, St. Mary's Ho@ital Medical School, London (Great Britain) Laboratory of Biology, National Cancer Institute, Bethesda, ;lid. (U.S.A.)
T. J. (7OLE~I.\x R. I). MARSH.XI.I. M. l)Ol"r E 1~:
I 91. POTTER, \V. J. DR~;VER, t-. I.. t(.L'FF AND l'~. R. M('[NTIRE, j. Mol. Bin/., 8 (19041 8i. I. z K. R. MclNTIRV AXD M. POTTI~R, J. Natl. Cancer Inst., 33 (I9641 o3I. 3 R. PERttAM, E. APPELLA AND .~l. POTTER, Science, 154 (I96¢1) 39I. 4 R. D..~IARSIIAI.I, AND M. POTTER, Abstr. 3rd ..1,Ieeting Federation t-uropean Biochem. Soc., H'arsau., I966, Academic Press, New York and P\VN, \Varsaw, 19()0, p. 258. 5 C. FRANq'OlS, R. D..MARSHALL ,~XD A. NEt:HERGER, Biochem. J., 83 (19~32) 335. O M. I1. (;IBt~ONS, Analyst, 8o (I9551 268. 7 J. (;- t,~RAA.NAND tl. Mt:IR, Hiochem. J., 66 (19571 55 I'. 8 l.. \VAgREX, J. Biol. Chem., 234 (I9591 I97 I. 9 F. MFLCHERS, I'~..S.I.ENNOX AND 31. FACON, Biochem. Biophys. Res. Commun., 24 (190o1 244. 1o 11. S. FRIEDMAX, Clin. Chim. Acla, 6 (1901) 775. 1I J. BoD,~.ax, Lab. Pratt., 0 (J957) 517. I2 S. MOORE AYD W. tl. STETS, .]. Biol. Chem., "21I (19541 9o7. x3 J. W. ROSEW.;.aR AXl) F. I.. SMITH, J. Biol. Chem., 23~) (1(1611 425. 14 C. NOLAN AND 1'2. L. SMITH, J. Biol. Chem. 237 (19112) 440, ,t5315 R. l)..MARSHALL AND A. NEUt~ERGER, in the press. I() A. (;OTTSCHAI. K, in A. (;OTTSCHAI.K, The Glycoprotein& t " l s e v i e r , Amsterdam, 1Oo(,, p. 282. 17 W. 1/. C-RAY, \V. J. ])REYER AND 1.. IIOOD, Science, 155 (t907) 405 . 18 A. NEUBF.RGER AND I/. l)..~,]ARSI4ALL, ill A. (;OTTSCIIALK, The (;lycoproteins, E l s e v i e r , A m s t e r dana, 1966, p. 230. 19 T. \V. (;OODWlX AXD R. A. MORTON, lliochem. ,]., ,to (194b) 628. 20 F..~IEI.CHI'LRS AND P. M. KNOPF, ill t h e p r e s s .
Received
July izth,
1967
Biochim. tliophy.s..4cla, I47 (x()(171 396--39,~
BIOCHI.~,tlCA I';T B I O P H Y S I C . \
PRELIMINARY
A{;TA
NOTES
BBA 3 I O I 7
Preparation of a glycopeptide from an immunoglobulin kappa polypeptide chain from the mouse We recently reported that a plasma cell t u m o u r (MOPC 4 6) which was induced in I~IALB/e micO -s secretes a kat)pa-type light chain with a relatively high cart)ohydrate content *. This is an unusual finding since light chains are generally associated with only small amtmnts of carbohydrate. In the present paper we describe further observations on this protein. In the first place we have analysed the sugar contents of proteins obtained from tumours at two different transfer generations (G 4 and Ga.0 and the results indicated that they were essentially the same (Table I). These data {lifter from those which were ret)orted t)y MELCHERS, I.F,CNNOXAND I?A('{)N 9 for a similar protein produced by an{}ther transplant line of the same tumour. We have also isolated :t glycopeptide from the protein. TAI~LE
I
CARBOHYDR.~.TI';
{:ON'rENT
TXVO G E N E R A T I O N S
T u r n o u t generalio~
134 {;a:,
OF
KAI'PA-TYPE
(}F TUMOUR
LIGIIT
{ tIAIN
PRO1)UtTEI)
BY MOUSE
TIJMOUR
.M()PC 4 6
A'F
TRANSPLANT
Carbohydrate content (g reh'ased per soo g prote~n) Hexose *
th, aosamine""
S i a l i c acid" * *
-t-5.t -t.3-'
2.88 2.74
o.9I o.85
* Mannose and galactose. The values were obtained by an orcinol-sulphuric a n d i n c l u d e a c o n t r i b u t i o n d u e t o t h e s m a l l a m o u n t of f u c o s e ~ (o.34 % ) p r e s e n t . * * Estimated as glucosamineL * * * M e a s u r e d a s N - a c e t y l n e u r a m i n i c a c i d ~.
acid method s
All the carbohydrate moved with the protein when tile latter was subjected to electrophoresis (pit 8.0) on cellulose acetate "). Moreover, if the protein were pretreated with 8 M urea, two protein bands of apt}roximately equal intensity appeared after electrt}phoresis, both of which stained with periodate-Schitt reagents alsolL These experiments SUpl)ort our belief that the carbohydrate which we have found occurs as an integral part of the light chain. The follmving procedure was used in order to isolate a glycopeptide from the protein (transplant generation 35). The glycoprotein (5o rag) was dissolved in 5 ml o.o5 M t)hosphate buffi:r, pH 7.5, which contained o.oi M EI)TA. bIercurit)apain (t rag; Sigma Laboratories, Lot 55 B;'I5OO) was added and the solution was maintained at 5 o~ and ptt 7.5. By following the course of the reaction with a t)ht}tometric nmhydrin method lz it was shown that r e a c t i o n was complete after about 2 h when a b o u t Hiochim. t¢iophys..-Iota, ]47 (x{) ~71 39{' 398
PREI.IMINARY
397
NOTES
25 O,.oof the peptide bonds of the protein had been cleaved. The pH of the digest was adjusted to 5.5:1.63 mg bound hexose were present at this stage. Ethanol (Io ml) was added to the solution and the small amount of precipitate which formed was removed by eentrifugation, leaving ~.32 nag hexose in the supernatant. The latter was taken to dryness i n vacuo and the residue extracted with successiw." i5-ml fractions of ethanol containing increasing amounts of water. The fraction e.xtracted with water-.ethanol (2o:8o, v/v) contained o.4 nag hexose and, judging by photometric ninhydrin assays, was least contaminated with peptides which did not contain carbohydrate. It contained about 2o moles of peptide lacking carbohwtrate for each mole of glyc opeptide. This fraction was further purified by chromatography on Dowex 5o-X8 (H~), with water as eluting agent Is,'4. About (}o % of the carbohydrate applied to the column was recovered. This glycopeptide was analysed by column chromatography after acid hydrolysis for its amino acid and amino sugar contents (Table II). It was also shown to "fA BIA'; II AMINO
ACID
LIGHT
CHAIN
AND
AMINO
PRODUCED
SUGAR
ANALYSIS
BY MOUSE
Component
Asl>artic acid * Serine Glutamic acid Glycine Isoleucine Glucosamine
OF
"FUblOUR
A (H.YCOPEPTIDE
ISOLATED
FROM
THE
KAPPA-TYPE
MOPC 40
Hydrolysis conditions 6 3I tlCI; 1zoO; I6 h
4 31 IIC); IOO°; 3 h
x .o o.97 o.88 o.38 o.38 2.o 3
i .o o.87 I. x 7 o.52 o.39 3.~
* Taken as standard.
contain 4.2 moles hexose per mole of aspartic acid. The glycopeptide gave rise to mannose and galactose on acid hydrolysis (2 M HC1; IOO~; 3 h), the former in slightly larger amounts. Small quantities of fucose were also shown to be present by paper chromatography (ethyl acetate-pyridine-water (12:5:4, by vol.)). The amino sugar was identified as glucosamine by the column chromatographic techniques employed. The nature of the carbohydrate-pel)tide bond in this protein has not been rigorously established yet but it is likely to be 2-acetamido-I-(fl-I.-aspartanfido)-i,2dideoxy-fl-D-glucose as in certain other glycoproteins Is. Thus treatment of the whole protein with o.~ M NaOH for 24 h at room temperature did not result in a splitting of the carbohydrate from the polypeptide chain. 2\ linkage involving the hydroxyl grou t) of serine is thus excluded 'n. Of the other amino acids identified in the glycopeptide only aspartic acid, or more correctly asparagine, is likely to provide the point of attachlnent of the carbohydrate moiety to the polypeptide chain, by analogy with other glycotlroteins. Moreover the only sugar yet shown to be involved in a linkage to asparagine in a glycoprotein is N-acetyl-l)-glucosamine'~. It is relevant to mention that a similar protein produced by tumour MOPC 41 has the sequence -Ser-Gln-Asx-Ile-Gly-- at positions 26-3o inclusiw~ froln the Ihoch¢m. t3iophys. Acta, Lt7 ( i 9 0 7 ) 3 9 6 - 3 9 8
398
I>RI.;I.I MI NAIC~" N() I'E5
N - t e r m i n a l e n d of t h e p o l y p e p t i d e c h a i n ' L It is ilot u n r e a s o n a b l e to s u g g e s t , f r o m a c o n s i d e r a t i o n of t h e a m i n o acid a n a l y s i s of o u r glycotw.ptide ( ' f a b l e II) t h a t 1he c a r b o h v d r a t c I n o i e t v w a s origin;ally a t t a c h e d a t a n asparagilu', r e s i d u e w h i c h ~)t'Cllrs in a s i m i l a r p - s i t | o n in t h e g l y c o p r o t r i n . T h e n o n - i n t c , ~ r a l \;titles f o u n d for l)t~tlt g l y c i l w a n d i s o l e u c i n c m o s t p r o b a b l y a r o s e b e c a u s e of p a r t i a l t'lc~tvage, b v t h e p a t ) a i n , ,)f t h e l i n k a g e b e t w e e n lie a n d t h e ASh r e s i d u e c a r r y i n g t h e c a r b o h y d r a t e m o i r t v . P a r t i a l c n z v m a t i c cle;tvage of l)t,ptide b o n d s in a n a l o g o u s p o s i t i o n s in o t h e r g l y c o p r o t c i n s
h~ts SOlllt'tinlcs ])e(!n ()])SCl\'t'd Is. It was m e n t i o n e d a b o v e t h ; t t a s i m i l a r p r o t e i n p r t ) d u c e d b y a n o t h e r t r a n s p l a n t line of the. s a m e t u r n o u t w a s also a glycoprotein but that it c o n t a i n e d m o r e carl)oh y d r a t e t h a n is p r e s e n t in . u r preparations..'~IEI.CHERS, LI-NNOX .\NI) FACO.X~ f o u n d t h a t t h e i r g l y c o p r o t c i n c t m l d be s p l i t i n t o t h r e e c o m p o n e n t s b y c h r o m a t o g r a p h y on I ) l ' . - \ E - e e l h f l o s e . T h e s e c o m p o n e n t s d i f f e r e d in t h e i r sialic acid c o n t e n t s w h i c h were s h o w n to b e a p l ) r o x , o, I, a n d 2 rcsi(lues rest)ectively'. F.ach (,f t h e s e c h r o m a t o g r a p h i c f r a c t i o n s w a s r e p o r t e d t o c o n t a i n a b o u t 4 r e s i d u e s e a c h (,f nl~tnll()S(! a n d of g a l a c t , s c , 2 of fucose an(l {) of h e x o s a m i m ' , a m o u n t s w h i c h a r e a b o u t t w i c e tll,~sc, w h i c h we lind in o u r proteins ( T a b l e I). I t s h o u l d also b e m e n t i o n e d t h a t t h e t r y p t o l ) h a n c o n t e n t w h i c h we h a v e f o u n d (3.5 residues) ~:' is l o w e r t h a n t h a t r e p o r t e d b y .MELCHH{S, LE.x'.x:C X ..X.','l) I:..\CoX" in t h e i r t h r e e f r a c t i o n s (5.4 to 5.6 residues). T h e r e a s o n s for t h e s e d i f f e r e n c e s a r e n o t v e t c]car. V e r y r e c e n t l y M1..I.cIH.;RS AND I(xt)l'l :e° h a v e r e p o r t e d t h e p r e p a r a t i o n . f a g l y c o p c p t i d e f r o m a t r y p t i c d i g e s t of t h e i r l i g h t c h a i n .
Department of ('heroical l'athology, St. Mary's Ho@ital Medical School, London (Great Britain) Laboratory of Biology, National Cancer Institute, Bethesda, ;lid. (U.S.A.)
T. J. (7OLE~I.\x R. I). MARSH.XI.I. M. l)Ol"r E 1~:
I 91. POTTER, \V. J. DR~;VER, t-. I.. t(.L'FF AND l'~. R. M('[NTIRE, j. Mol. Bin/., 8 (19041 8i. I. z K. R. MclNTIRV AXD M. POTTI~R, J. Natl. Cancer Inst., 33 (I9641 o3I. 3 R. PERttAM, E. APPELLA AND .~l. POTTER, Science, 154 (I96¢1) 39I. 4 R. D..~IARSIIAI.I, AND M. POTTER, Abstr. 3rd ..1,Ieeting Federation t-uropean Biochem. Soc., H'arsau., I966, Academic Press, New York and P\VN, \Varsaw, 19()0, p. 258. 5 C. FRANq'OlS, R. D..MARSHALL ,~XD A. NEt:HERGER, Biochem. J., 83 (19~32) 335. O M. I1. (;IBt~ONS, Analyst, 8o (I9551 268. 7 J. (;- t,~RAA.NAND tl. Mt:IR, Hiochem. J., 66 (19571 55 I'. 8 l.. \VAgREX, J. Biol. Chem., 234 (I9591 I97 I. 9 F. MFLCHERS, I'~..S.I.ENNOX AND 31. FACON, Biochem. Biophys. Res. Commun., 24 (190o1 244. 1o 11. S. FRIEDMAX, Clin. Chim. Acla, 6 (1901) 775. 1I J. BoD,~.ax, Lab. Pratt., 0 (J957) 517. I2 S. MOORE AYD W. tl. STETS, .]. Biol. Chem., "21I (19541 9o7. x3 J. W. ROSEW.;.aR AXl) F. I.. SMITH, J. Biol. Chem., 23~) (1(1611 425. 14 C. NOLAN AND 1'2. L. SMITH, J. Biol. Chem. 237 (19112) 440, ,t5315 R. l)..MARSHALL AND A. NEUt~ERGER, in the press. I() A. (;OTTSCHAI. K, in A. (;OTTSCHAI.K, The Glycoprotein& t " l s e v i e r , Amsterdam, 1Oo(,, p. 282. 17 W. 1/. C-RAY, \V. J. ])REYER AND 1.. IIOOD, Science, 155 (t907) 405 . 18 A. NEUBF.RGER AND I/. l)..~,]ARSI4ALL, ill A. (;OTTSCIIALK, The (;lycoproteins, E l s e v i e r , A m s t e r dana, 1966, p. 230. 19 T. \V. (;OODWlX AXD R. A. MORTON, lliochem. ,]., ,to (194b) 628. 20 F..~IEI.CHI'LRS AND P. M. KNOPF, ill t h e p r e s s .
Received
July izth,
1967
Biochim. tliophy.s..4cla, I47 (x()(171 396--39,~