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Detection of unique peptides derived from the μ chains of cell surface but not secreted IgM
Moleculur Printed
hli?lUW/O<~J~.
in Great
Vol.
18, No. 6, pp. 467471,
198 I.
0161-5890,‘81;‘060467-05f02.00/0 Pergamon Press Ltd.
Britam
DETECTION OF UNIQUE PEPTIDES DERIVED FROM THE p CHAINS OF CELL SURFACE BUT NOT SECRETED IgM* DOROTHY
YUAN,
Dcpartmcnt of Microbiology.
JONATHAN
University
W. UHR
and ELLEN
of Texas Southwestern U.S.A.
Medical
S. VITETTA School,
Dallas. TX 75235.
(Received 24 Seprtlmber 1980; awepied 12 Nowmbrr 1980) Abstract
IgM is present on the surface of B lymphocytes in monomeric form and can also be secreted into the serum in pentamcric form after differentiation of B cells into plasma cells. We have previously used a monoclonal population of murine tumor cells (BCL,) which express surface IgM but can also secrete IgM after stimulation with lipopolysaccharide to study the primary structure of bl chains from ccl1 surface and secreted IgM. Using cation exchange chromatography, we detected a peptide prcscnt only in 1, chains from secreted IgM but not in cell surface IgM. The putative extra peptide from the cell surface I, chain could not be identified perhaps due to its inability to bind to the exchange resin. In the prcsznt study we have used reverse phase high pressure liquid chromatography to analyse the peptides of/r chains from cell surface and secreted IgM from both BCL, tumor cells and from normal B lymphocytes. Our results demonstrate the presence of two peptides associated with the Fc portion of the cell surface but not with the secreted ,n chain.
INTRODUCTION
extra peptide was present only in I( chains from secreted IgM, The putative extra peptidz(s) from the ceil surface I’ chain could not be identified perhaps due to its inability to bind to the exchange resin. In the present study, we have analysed the peptides of jl chains from cell surface and secreted IgM using reverse phase high pressure liquid chromatography (HPLC). This technique separates peptidcs on the basis of hydrophobicity (Molnar & Horvath. 1977). Our results demonstrate the presence of two peptidcs associated with the Fc portion of the cell surface but not with the secreted p chain.
IgM is present on the surface of B lymphocytes in monomeric form (/ILLS) (Vitetta et a/.,1971) and can also be secreted into the serum in pentameric form (/t, ,L, 0) after differentiation of B cells into plasma cells. The /J chains of cell surface IgM differ from those of secreted IgM in their apparent molecular weight (Melcher & Uhr, 1973: Vassalli c’t ol., 1979). solubility (M&her et trl.. 1976). detergent binding (Vassalli et (II., 1979: Parkhouse ct ol.. 1980) and buoyant density (M&her & Uhr. 1977). These differences suggest that I/ chains from cell surface IgM have an extra hydrophobic sequence responsible for anchoring the molecules in the plasma membrane (Vitetta & Uhr. 1973). We have previously used a monoclonal population of murine tumor cells (BCL,) (Slavin & Strober, 1977) which express surface IgM but can also secrete IgM after stimulation with lipopolysaccharide (LPS) to study the primary structure of cl chains from cell surface and secreted IgM (Yuan et LIP.,1980). The results indicated only one peptide difference between the tryptic-chymotryptic peptides of the two I/ chains. These analyses were performed using cation exchange chromatography and the
*Supported by NIH Grants
AI-12789,
AI-11851.
MATERIAIS
BCL,
AND METHODS
crlls
BCL, cells were maintained in BALB/c mice (Vitetta er al., 1979) obtained from Cumberland Farms, Clinton. TN. Preptrrution
qf’ r~rtlioltrheled
11 chuins
Spleen cells from normal or tumor-bearing mice were labeled in vitro with either 3H- or i4Ctyrosine for 8 hr and lysed in 0.5”; Nonidet P40 (NP40) (Yuan et r/l., 1980). Lysates were chromatographed on a Sepharose-lentil lectin affinity column to obtain the glycoprotein pool (GPP). To prepare secreted IgM, BCL, cells or BALB/c splenocytes were cultured for two days
and
CA-23115. Abhrev/rrtion.r LPS, lipopolysaccharide: HPCL. high pressure liquid chromatography: NP40. Nonidet P40: GPP. glycoprotcin pool: SDS. sodium dodecyl sulfate: PAGE. polyacrylamide gel electrophoresis. 467
with LPS (Yuan c’f if/.. 1980). harvcstzd and labzlzd with tyrosinz for I6 hr. IgM was isolated from czll lysatcs and sc:crctionh by binding it to rabbit anti-mouse: i. followed by adsorption of immune complzxcs to S. 01ir(v/\ (Vitztta (‘f rfl.. 1979). The immune: complcxc\ \\crc c:luted in 8 M ur2a containing sodium dodccyl sulfate (SDS) and the L/ chains w,.:r%: rl:soIvt:d by SDSpolyacrylamidc (SDSg21 clcctrophorzsis PAGE).
Th(: GPP from ‘SC-tyrosinc lab&d BCL, cells was treated with rabbit anti-mouse I; (Vitetta Ed111..1979) and S. ~IIII’CIIS to rcmovI: IgM from contaminating normal lymphoid cells. The remaining i-containing 1gM was then isolated by binding it to rabbit anti-MOPC-104E (Isakson cf ol.. 1980). Thi: immune: complcxzs wt:ri: digcstcd while still adsorbed to S. IIIIIX’LIS\vith TPCK trypsin (Worthington Biochcmicals. Fr~hold. NJ, 75 j/g pzr ml of lo”,, S. (1~4~~~~.\ susp,:nsion) at 56 C‘ for 30 min in 0.04 2/1 Tris HCI. 0.005 I4 CaCl?. pH X.0. The: Fc fragments and undigested 1’ chain which rtimain attached to S. LIIIR’CIS wcrc elut;:d with SDS and r.:solvcd by SDS-PAGE. Undk:r non-reducing conditions. th,: F‘c(/l) miprat,:d with an appar;:nt mol::cular wi:ight of
50
-_. 100 FRACTIONS
102.000 d. After ri:ducrlon. ;I \ingl< of 54.000 d wah obtain2ci.
moi~culal
sp~cii's
“H- or 15C-labeled ~1chains from cell associated and secreted IgM were mixed and digested together with chymotrypsin followed by trypsin (Yuan ct t/l., 1980). Dig&ion products wcrc lyophilizcd and dissolved in 7”,, actitic acid. HPLC was carried oul in a Beckman Gradient Liquid Chromatograph. Model 332. The concentration of the organic phase: (acetonitrilc. Burdick and Jackson Labs, Muskegon. MI ) u as incrcaszd to 25”,, within a period of 60 min. 0.3 ml fractions wcrc collected and counted in Beckman H/P cocktail. “H- and ‘“C-cpm wcrt‘ normal&d and corrected for channel spillovcrs.
HESC 1:l.s
Using IgM from BCL, cells labeled with “H- or ‘jC-tyrosine, the chymotryptic-tryptic peptidcs of jt chains from ccl1 associated and secreted IgM wt’rc cochromatographcd by H PLC. Figure 1A sho\vs that the peptides from
150
200
Fig. I. HPLC of chymotryptic-tryptic digests of p chains prepared from the IgM of the BCL, tumor. (a) 3H-tyrosine labeled ~1 chains from cell associated IgM were digested and co-chromatographed with ‘“C-labeled p chains from secreted IgM (b) jH-tyrosine labeled 11 chains from cell associated IgM were digested and co-chromatographed with 14C-tyrosine labeled p chains from the same source. Arrows indicate the reproducible differences found in A but not 9. The stippled peak represents the unique-secreted peptide and the striped peaks the unique surface peptides.
Unique
Peptides
in 11Chain from Cell Surface
50
IgM
469
150
100 FRACTIONS
Fig. 2. HPLC of chymotryptic-tryptic digests of Fc fragments of kt chains prepared from the IgM of the BCL, tumor. Fe(p) fragments derived from cell associated IgM labeled with 14C-tyrosine were co-chromatographed with intact p chains derived from secreted IgM labeled with 3H-tyrosine. Arrows indicate positions of putative Fd peptides.
the two sources co-elute with 4 exceptions: at 16:~ acetonitrile (fraction 115), a unique peptide derived from the cl chains of secreted IgM is present. In addition, 3 unique peptides in the /I chains of cell associated IgM elute with 1490 (fraction loo), 227; (fraction 140), and 25”,, (fraction 160). With the exception of the peptide eluted at 259; acetonitrile, the above differences were not seen when “H- and 14C-tyrosine labeled L/chains from cell ussociated IgM were compared (Fig. 18). Thus, the peptide eluted at 25”; acetonitrile represents a technical artifact rather than a peptide difference. In a total of ten experiments utilizing double labels, we have observed the unique secreted peptide tight times and both of the unique cell associated peptides seven times. The failure to observe the unique peptides in some experiments was due to poor resolution of the peptides. The above results were obtained with cell which include both associated ,U chains intracellular and cell surface ~1chains (Yuan et
50
100 FRACTIONS
u/., 1980). These 2 forms can be partially resolved on SDS-PAGE because of differences in extent of glycosylation (Yuan et al., 1980). Thus, only the slowly migrating il chains are radiolabeled by enzymatic radioiodination or bound by anti-i on intact BCL, cells (Yuan et al., 1980). To study the peptides from cell surface /I chains, we pooled fractions from the more slowly migrating cell associated p chains. These were cochromatographed with the chymotryptic-tryptic peptides prepared from the /I chains of secreted IgM. The map (not shown) indicates that the two peptides unique to kl chains from cell associated IgM are also present in /r chains of cell surjke IgM. 7he
unique
lrssocitrted molecule
peptides
from
1gM trre locutetl
,LI chains in the Fc portion
of‘ cell of’the
Fc(kr) fragments from cell associated IgM were prepared and digested together with intact !C chains from secreted IgM. As denoted by the
150
Fig. 3. HPLC of chymotryptic-tryptic digests of p chains from IgM derived from BALB/c splenocytes. “H-tyrosine labeled kt chains derived from cell associated IgM of BALB/c splenocytes were digested and co-chromatographed with ‘%-tyrosine labeled /r chains from IgM secreted by LPS-stimulated BALB/c splenocytes.
arro\\
in
5
I- ig.
pr~:sumably
3.
p,:ptitii:s
associarcd
I I”,,
in
th.,
pi:plitlA
portion
01’ rh
: 11
I+c.(/I 1 p~~~~i~~n ()I‘ c(:ll
;tri! p~-,:~n(
[fraclion
of
l-d
Fc(~I 1. Ffo\\.:\C:r. lh<, I\\0
from
IfM
Illlnlb;:r
in lh,:
chains ar2 absent uniqu,:
:I
located
X0]
(fraction\
and
,:lut illy
[ fraction
2.3”
at
1-W)
acctonitril.:). nW I/!fi~jlW fum
(,?/I
[“‘~Jfii/C.\
(/I’(’
r/c.\oc,iofd
c//.\O /JW\C,11
lq.W
o/~roirl~~t/
i/f j’
JWIII
C/Jr/ifJ\
,lo/~r,rc//
H
W/l.\
chains
!l
da-ivi:d secrctcd
by
(Fig.
p~ptidcs BCL,
bilh
BALB/c 3)
indicata in
[fraction
11 chains thal
c::ll
origin
in
2 arc
,I/?
t/U Ir,liql”’
To
dr:1;:rmini: BCL
‘H-gluc()S;lmin
dig,:\l,.:d \\ith “C‘-lal~:l.:ti 2 uniqu::
surface
resulting
IgM.
ac.:lonitrili~ I I
l7”,,
th,:
glycopi:ptidC:
111,: pi:ptida
from
display
;I
p.:ptidza
obtain;:d
22
\li-uctural
2?“,,
:lut .:d al
r.:soh.:d
I‘I-om
p,:ak\. ‘I hu\. at I::art
from
that ~)fc:ll
and
1h ’ on2
cull as\ociat:d
primary
\,:ci-.:t,:d
indicat.:s
he~ween
is not glycc~\ylat :ti:
chain\
I/
in j/chains
co~ild ii01 b,: d,:linili~,:l~
putativ,:
01
IgM
:. -fuco\G:. anti and
pra..:nt
eluting
diff,:r
:tssociat,:d
chromatogram
one
p~.:ptid.:\ IgM
J/ chain\ from
p,:piiti~s the
IgM.
IgM by \ irru.-
c::ll
ef cl/.. 19x0)
Th,:
as
this map
uniqu,:
from s;:cr>:tzd
-galactos.: IgM.
from
th,:
\V;IS l>~b~l~d with
oftli.‘
IRON]
di:signafai
associatai
glycosylalion. (Yuan
of
I7”,,
at
(//VC.O.\ I./~//CJ”
whL:ihi:r
from thos,: d,:rivi:d
from lgh4
[fraclion
akxn~
l I/ chains of all
diff,:r.:ntial
from
of polyclonal
pq’ritle
L~niqLi,:
IyM
.:luting
22”,,
the h&a-ogaii:ity
to
from
(fractions
IgM
pqtiti~
1~0
of the p~ptid~s
in Eig.
~1~1:
lhi:
as\oci:~tzti
and
Many
1gM \\:.:r,:
from TIC
in /f chains
c~:Ils
1201
aci:tonitrilc).
;I\\ociat&,ti \pli:nocyt,:s
splznocyta.
pre+:nt splzi:n
ct:ll
BALB/c
cells arc: przscnt
normal
F‘d
from
normal
togcthcr
dip:slxi
map
prcparai
from
oiii’
01
11 chain\ must tiif12ri:nc6:
11chain5 ofs_:crr:l
from
:tl IgM.
l~ISwSSIoN The
of
present
studies
using
trypticechymotryptic
the HPLC’
digests
analysis
have detected
three peptide differences
between 14 chains
surface and secreted IgM
: two of the peptides art:
unique
to or chains
results
extend
structural ~l chains Singer known
of cell surface
previous
differences (Yuan
IgM.
Thcsc
of primary
between these two typt’s of
el L/I..
rt al., 1980)
observations
of cell
1980: Williams
and are consistent
biochemical
differences
ef (I/..
19’78:
with
their
and
the
Umque
Peptides
in /r Chain
Jaton J. C. & Vassalli P. (1980) A major structural difference between membrane-bound and secreted IgM of normal mouse spleen cells is located in the C-terminal region of their heavy chams. FEBS Lrrtrrs 116, 277. Kehry M., Sibley C., Fuhrman J., Schilling J. & Hood L. E. (1979) Amino acid sequence of a mouse immunoglobulin ir chain. Proc. nrrtn. Arud. Sri. U.S.A. 76, 2932. McCune J. M., Lingappa V. R., Fu S. M., Blobel G. & Kunkel H. G. (1980). Biogenesis of membrane-bound and secreted immunoglobulins. I. Two distinct translation products of human 1’ chains. with identical N-termini and different Ctermini. J. Exp. Med. 152, 463. Melchcr U.. Eidels L. & Uhr J. W. (1976) Are Igs membrane proteins? Naturr, Land. 258, 434. Melcher U. & Uhr J. W. (1973). An electrophoretic difference between surface and secreted IgM of murine splenocytes. .I. Exp. Med. 138, 1282. Melcher U. & Uhr J. W. (1977) Density differences between membrane and secreted immunoglobulins of murine splenocytcs. Biochemistry 16, 145. Molnar I. & Horvath C. (1977) Separation of amino acids and peptides on non-polar stationary phases by highperformance liquid chromatography. J. Chromutogruph~ 142, 623. Parkhouse R. M. E., Lifter J. & Choi Y. S. (1980) Chemical characterization of the Fab and Fc fragments from surface immunoglobulin. Nature, Land. 284, 280. Rogers J., Early P., Carter C., Calame K., Bond M., Hood L. & Wall R. (1980) Two mRNAs with different 3’ends encode
from Cell Surface
IgM
471
membrane-bound and secreted forms ofimmunoglobulin 1’ chain. Cell 20, 303. Singer P. A., Singer H. H. & Williamson A. R. (1980) Different species of messenger RNA encode receptor and secretory IgM jr chains differing at their carboxy tcrmini. Nature, Lend. 285, 294. Slavin S. & Strober S. (1977) Spontaneous murtne B cell leukemia. N~ltrw. Land. 272, 624. Vassalli P., Tedghi R., Lisowska-Bernstein B., Tartakoff A. & Jaton J. C. (1979) Evidence for hydrophobic region with heavy chains of mouse B lymphocyte membrane-hound IgM. Pnx. nurn. Amd. %i. U.S.A. 76, 5515. Vitetta E. S., Baur S. & Uhr J. W. (1971). Cell surfacc Ig.11. Isolation and characterization of Ig from mouse splenic lymphocytes. J. Exp. Med. 134, 242. Vitetta E. S. & Uhr J. W. (1973) Synthesis. transport, dynamics, and fate of cell surface Ig and alloantigens in murine lymphocytes. Transplunr. Rec. 14, 50. Vitetta E. S., Yuan D., Krolick K., Isakson P., Knapp M., Slavin S. & Strober S. (1979) Characterization ot a spontaneous murine B cell leukemia (BCL, ). III. Evidence for monoclonality using anti-idiotypic antibody. J. Immunol. 122, 1649. Williams P. B.. Kubo R. T. & Grey H. M. (1978) Ii-Chains from a nonsecretor B cell line differ from secreted Iwzhains at the C-terminal end. J. Immunol. 121, 2435. Yuan D.. Uhr J. W. & Vitetta E. S. (1980) A peptidediffcrcnce between /t chains from cell associated and secreted IgM of the BCL, tumor. J. Inznzunol. 125, 40.
hli?lUW/O<~J~.
in Great
Vol.
18, No. 6, pp. 467471,
198 I.
0161-5890,‘81;‘060467-05f02.00/0 Pergamon Press Ltd.
Britam
DETECTION OF UNIQUE PEPTIDES DERIVED FROM THE p CHAINS OF CELL SURFACE BUT NOT SECRETED IgM* DOROTHY
YUAN,
Dcpartmcnt of Microbiology.
JONATHAN
University
W. UHR
and ELLEN
of Texas Southwestern U.S.A.
Medical
S. VITETTA School,
Dallas. TX 75235.
(Received 24 Seprtlmber 1980; awepied 12 Nowmbrr 1980) Abstract
IgM is present on the surface of B lymphocytes in monomeric form and can also be secreted into the serum in pentamcric form after differentiation of B cells into plasma cells. We have previously used a monoclonal population of murine tumor cells (BCL,) which express surface IgM but can also secrete IgM after stimulation with lipopolysaccharide to study the primary structure of bl chains from ccl1 surface and secreted IgM. Using cation exchange chromatography, we detected a peptide prcscnt only in 1, chains from secreted IgM but not in cell surface IgM. The putative extra peptide from the cell surface I, chain could not be identified perhaps due to its inability to bind to the exchange resin. In the prcsznt study we have used reverse phase high pressure liquid chromatography to analyse the peptides of/r chains from cell surface and secreted IgM from both BCL, tumor cells and from normal B lymphocytes. Our results demonstrate the presence of two peptides associated with the Fc portion of the cell surface but not with the secreted ,n chain.
INTRODUCTION
extra peptide was present only in I( chains from secreted IgM, The putative extra peptidz(s) from the ceil surface I’ chain could not be identified perhaps due to its inability to bind to the exchange resin. In the present study, we have analysed the peptides of jl chains from cell surface and secreted IgM using reverse phase high pressure liquid chromatography (HPLC). This technique separates peptidcs on the basis of hydrophobicity (Molnar & Horvath. 1977). Our results demonstrate the presence of two peptidcs associated with the Fc portion of the cell surface but not with the secreted p chain.
IgM is present on the surface of B lymphocytes in monomeric form (/ILLS) (Vitetta et a/.,1971) and can also be secreted into the serum in pentameric form (/t, ,L, 0) after differentiation of B cells into plasma cells. The /J chains of cell surface IgM differ from those of secreted IgM in their apparent molecular weight (Melcher & Uhr, 1973: Vassalli c’t ol., 1979). solubility (M&her et trl.. 1976). detergent binding (Vassalli et (II., 1979: Parkhouse ct ol.. 1980) and buoyant density (M&her & Uhr. 1977). These differences suggest that I/ chains from cell surface IgM have an extra hydrophobic sequence responsible for anchoring the molecules in the plasma membrane (Vitetta & Uhr. 1973). We have previously used a monoclonal population of murine tumor cells (BCL,) (Slavin & Strober, 1977) which express surface IgM but can also secrete IgM after stimulation with lipopolysaccharide (LPS) to study the primary structure of cl chains from cell surface and secreted IgM (Yuan et LIP.,1980). The results indicated only one peptide difference between the tryptic-chymotryptic peptides of the two I/ chains. These analyses were performed using cation exchange chromatography and the
*Supported by NIH Grants
AI-12789,
AI-11851.
MATERIAIS
BCL,
AND METHODS
crlls
BCL, cells were maintained in BALB/c mice (Vitetta er al., 1979) obtained from Cumberland Farms, Clinton. TN. Preptrrution
qf’ r~rtlioltrheled
11 chuins
Spleen cells from normal or tumor-bearing mice were labeled in vitro with either 3H- or i4Ctyrosine for 8 hr and lysed in 0.5”; Nonidet P40 (NP40) (Yuan et r/l., 1980). Lysates were chromatographed on a Sepharose-lentil lectin affinity column to obtain the glycoprotein pool (GPP). To prepare secreted IgM, BCL, cells or BALB/c splenocytes were cultured for two days
and
CA-23115. Abhrev/rrtion.r LPS, lipopolysaccharide: HPCL. high pressure liquid chromatography: NP40. Nonidet P40: GPP. glycoprotcin pool: SDS. sodium dodecyl sulfate: PAGE. polyacrylamide gel electrophoresis. 467
with LPS (Yuan c’f if/.. 1980). harvcstzd and labzlzd with tyrosinz for I6 hr. IgM was isolated from czll lysatcs and sc:crctionh by binding it to rabbit anti-mouse: i. followed by adsorption of immune complzxcs to S. 01ir(v/\ (Vitztta (‘f rfl.. 1979). The immune: complcxc\ \\crc c:luted in 8 M ur2a containing sodium dodccyl sulfate (SDS) and the L/ chains w,.:r%: rl:soIvt:d by SDSpolyacrylamidc (SDSg21 clcctrophorzsis PAGE).
Th(: GPP from ‘SC-tyrosinc lab&d BCL, cells was treated with rabbit anti-mouse I; (Vitetta Ed111..1979) and S. ~IIII’CIIS to rcmovI: IgM from contaminating normal lymphoid cells. The remaining i-containing 1gM was then isolated by binding it to rabbit anti-MOPC-104E (Isakson cf ol.. 1980). Thi: immune: complcxzs wt:ri: digcstcd while still adsorbed to S. IIIIIX’LIS\vith TPCK trypsin (Worthington Biochcmicals. Fr~hold. NJ, 75 j/g pzr ml of lo”,, S. (1~4~~~~.\ susp,:nsion) at 56 C‘ for 30 min in 0.04 2/1 Tris HCI. 0.005 I4 CaCl?. pH X.0. The: Fc fragments and undigested 1’ chain which rtimain attached to S. LIIIR’CIS wcrc elut;:d with SDS and r.:solvcd by SDS-PAGE. Undk:r non-reducing conditions. th,: F‘c(/l) miprat,:d with an appar;:nt mol::cular wi:ight of
50
-_. 100 FRACTIONS
102.000 d. After ri:ducrlon. ;I \ingl< of 54.000 d wah obtain2ci.
moi~culal
sp~cii's
“H- or 15C-labeled ~1chains from cell associated and secreted IgM were mixed and digested together with chymotrypsin followed by trypsin (Yuan ct t/l., 1980). Dig&ion products wcrc lyophilizcd and dissolved in 7”,, actitic acid. HPLC was carried oul in a Beckman Gradient Liquid Chromatograph. Model 332. The concentration of the organic phase: (acetonitrilc. Burdick and Jackson Labs, Muskegon. MI ) u as incrcaszd to 25”,, within a period of 60 min. 0.3 ml fractions wcrc collected and counted in Beckman H/P cocktail. “H- and ‘“C-cpm wcrt‘ normal&d and corrected for channel spillovcrs.
HESC 1:l.s
Using IgM from BCL, cells labeled with “H- or ‘jC-tyrosine, the chymotryptic-tryptic peptidcs of jt chains from ccl1 associated and secreted IgM wt’rc cochromatographcd by H PLC. Figure 1A sho\vs that the peptides from
150
200
Fig. I. HPLC of chymotryptic-tryptic digests of p chains prepared from the IgM of the BCL, tumor. (a) 3H-tyrosine labeled ~1 chains from cell associated IgM were digested and co-chromatographed with ‘“C-labeled p chains from secreted IgM (b) jH-tyrosine labeled 11 chains from cell associated IgM were digested and co-chromatographed with 14C-tyrosine labeled p chains from the same source. Arrows indicate the reproducible differences found in A but not 9. The stippled peak represents the unique-secreted peptide and the striped peaks the unique surface peptides.
Unique
Peptides
in 11Chain from Cell Surface
50
IgM
469
150
100 FRACTIONS
Fig. 2. HPLC of chymotryptic-tryptic digests of Fc fragments of kt chains prepared from the IgM of the BCL, tumor. Fe(p) fragments derived from cell associated IgM labeled with 14C-tyrosine were co-chromatographed with intact p chains derived from secreted IgM labeled with 3H-tyrosine. Arrows indicate positions of putative Fd peptides.
the two sources co-elute with 4 exceptions: at 16:~ acetonitrile (fraction 115), a unique peptide derived from the cl chains of secreted IgM is present. In addition, 3 unique peptides in the /I chains of cell associated IgM elute with 1490 (fraction loo), 227; (fraction 140), and 25”,, (fraction 160). With the exception of the peptide eluted at 259; acetonitrile, the above differences were not seen when “H- and 14C-tyrosine labeled L/chains from cell ussociated IgM were compared (Fig. 18). Thus, the peptide eluted at 25”; acetonitrile represents a technical artifact rather than a peptide difference. In a total of ten experiments utilizing double labels, we have observed the unique secreted peptide tight times and both of the unique cell associated peptides seven times. The failure to observe the unique peptides in some experiments was due to poor resolution of the peptides. The above results were obtained with cell which include both associated ,U chains intracellular and cell surface ~1chains (Yuan et
50
100 FRACTIONS
u/., 1980). These 2 forms can be partially resolved on SDS-PAGE because of differences in extent of glycosylation (Yuan et al., 1980). Thus, only the slowly migrating il chains are radiolabeled by enzymatic radioiodination or bound by anti-i on intact BCL, cells (Yuan et al., 1980). To study the peptides from cell surface /I chains, we pooled fractions from the more slowly migrating cell associated p chains. These were cochromatographed with the chymotryptic-tryptic peptides prepared from the /I chains of secreted IgM. The map (not shown) indicates that the two peptides unique to kl chains from cell associated IgM are also present in /r chains of cell surjke IgM. 7he
unique
lrssocitrted molecule
peptides
from
1gM trre locutetl
,LI chains in the Fc portion
of‘ cell of’the
Fc(kr) fragments from cell associated IgM were prepared and digested together with intact !C chains from secreted IgM. As denoted by the
150
Fig. 3. HPLC of chymotryptic-tryptic digests of p chains from IgM derived from BALB/c splenocytes. “H-tyrosine labeled kt chains derived from cell associated IgM of BALB/c splenocytes were digested and co-chromatographed with ‘%-tyrosine labeled /r chains from IgM secreted by LPS-stimulated BALB/c splenocytes.
arro\\
in
5
I- ig.
pr~:sumably
3.
p,:ptitii:s
associarcd
I I”,,
in
th.,
pi:plitlA
portion
01’ rh
: 11
I+c.(/I 1 p~~~~i~~n ()I‘ c(:ll
;tri! p~-,:~n(
[fraclion
of
l-d
Fc(~I 1. Ffo\\.:\C:r. lh<, I\\0
from
IfM
Illlnlb;:r
in lh,:
chains ar2 absent uniqu,:
:I
located
X0]
(fraction\
and
,:lut illy
[ fraction
2.3”
at
1-W)
acctonitril.:). nW I/!fi~jlW fum
(,?/I
[“‘~Jfii/C.\
(/I’(’
r/c.\oc,iofd
c//.\O /JW\C,11
lq.W
o/~roirl~~t/
i/f j’
JWIII
C/Jr/ifJ\
,lo/~r,rc//
H
W/l.\
chains
!l
da-ivi:d secrctcd
by
(Fig.
p~ptidcs BCL,
bilh
BALB/c 3)
indicata in
[fraction
11 chains thal
c::ll
origin
in
2 arc
,I/?
t/U Ir,liql”’
To
dr:1;:rmini: BCL
‘H-gluc()S;lmin
dig,:\l,.:d \\ith “C‘-lal~:l.:ti 2 uniqu::
surface
resulting
IgM.
ac.:lonitrili~ I I
l7”,,
th,:
glycopi:ptidC:
111,: pi:ptida
from
display
;I
p.:ptidza
obtain;:d
22
\li-uctural
2?“,,
:lut .:d al
r.:soh.:d
I‘I-om
p,:ak\. ‘I hu\. at I::art
from
that ~)fc:ll
and
1h ’ on2
cull as\ociat:d
primary
\,:ci-.:t,:d
indicat.:s
he~ween
is not glycc~\ylat :ti:
chain\
I/
in j/chains
co~ild ii01 b,: d,:linili~,:l~
putativ,:
01
IgM
:. -fuco\G:. anti and
pra..:nt
eluting
diff,:r
:tssociat,:d
chromatogram
one
p~.:ptid.:\ IgM
J/ chain\ from
p,:piiti~s the
IgM.
IgM by \ irru.-
c::ll
ef cl/.. 19x0)
Th,:
as
this map
uniqu,:
from s;:cr>:tzd
-galactos.: IgM.
from
th,:
\V;IS l>~b~l~d with
oftli.‘
IRON]
di:signafai
associatai
glycosylalion. (Yuan
of
I7”,,
at
(//VC.O.\ I./~//CJ”
whL:ihi:r
from thos,: d,:rivi:d
from lgh4
[fraclion
akxn~
l I/ chains of all
diff,:r.:ntial
from
of polyclonal
pq’ritle
L~niqLi,:
IyM
.:luting
22”,,
the h&a-ogaii:ity
to
from
(fractions
IgM
pqtiti~
1~0
of the p~ptid~s
in Eig.
~1~1:
lhi:
as\oci:~tzti
and
Many
1gM \\:.:r,:
from TIC
in /f chains
c~:Ils
1201
aci:tonitrilc).
;I\\ociat&,ti \pli:nocyt,:s
splznocyta.
pre+:nt splzi:n
ct:ll
BALB/c
cells arc: przscnt
normal
F‘d
from
normal
togcthcr
dip:slxi
map
prcparai
from
oiii’
01
11 chain\ must tiif12ri:nc6:
11chain5 ofs_:crr:l
from
:tl IgM.
l~ISwSSIoN The
of
present
studies
using
trypticechymotryptic
the HPLC’
digests
analysis
have detected
three peptide differences
between 14 chains
surface and secreted IgM
: two of the peptides art:
unique
to or chains
results
extend
structural ~l chains Singer known
of cell surface
previous
differences (Yuan
IgM.
Thcsc
of primary
between these two typt’s of
el L/I..
rt al., 1980)
observations
of cell
1980: Williams
and are consistent
biochemical
differences
ef (I/..
19’78:
with
their
and
the
Umque
Peptides
in /r Chain
Jaton J. C. & Vassalli P. (1980) A major structural difference between membrane-bound and secreted IgM of normal mouse spleen cells is located in the C-terminal region of their heavy chams. FEBS Lrrtrrs 116, 277. Kehry M., Sibley C., Fuhrman J., Schilling J. & Hood L. E. (1979) Amino acid sequence of a mouse immunoglobulin ir chain. Proc. nrrtn. Arud. Sri. U.S.A. 76, 2932. McCune J. M., Lingappa V. R., Fu S. M., Blobel G. & Kunkel H. G. (1980). Biogenesis of membrane-bound and secreted immunoglobulins. I. Two distinct translation products of human 1’ chains. with identical N-termini and different Ctermini. J. Exp. Med. 152, 463. Melchcr U.. Eidels L. & Uhr J. W. (1976) Are Igs membrane proteins? Naturr, Land. 258, 434. Melcher U. & Uhr J. W. (1973). An electrophoretic difference between surface and secreted IgM of murine splenocytes. .I. Exp. Med. 138, 1282. Melcher U. & Uhr J. W. (1977) Density differences between membrane and secreted immunoglobulins of murine splenocytcs. Biochemistry 16, 145. Molnar I. & Horvath C. (1977) Separation of amino acids and peptides on non-polar stationary phases by highperformance liquid chromatography. J. Chromutogruph~ 142, 623. Parkhouse R. M. E., Lifter J. & Choi Y. S. (1980) Chemical characterization of the Fab and Fc fragments from surface immunoglobulin. Nature, Land. 284, 280. Rogers J., Early P., Carter C., Calame K., Bond M., Hood L. & Wall R. (1980) Two mRNAs with different 3’ends encode
from Cell Surface
IgM
471
membrane-bound and secreted forms ofimmunoglobulin 1’ chain. Cell 20, 303. Singer P. A., Singer H. H. & Williamson A. R. (1980) Different species of messenger RNA encode receptor and secretory IgM jr chains differing at their carboxy tcrmini. Nature, Lend. 285, 294. Slavin S. & Strober S. (1977) Spontaneous murtne B cell leukemia. N~ltrw. Land. 272, 624. Vassalli P., Tedghi R., Lisowska-Bernstein B., Tartakoff A. & Jaton J. C. (1979) Evidence for hydrophobic region with heavy chains of mouse B lymphocyte membrane-hound IgM. Pnx. nurn. Amd. %i. U.S.A. 76, 5515. Vitetta E. S., Baur S. & Uhr J. W. (1971). Cell surfacc Ig.11. Isolation and characterization of Ig from mouse splenic lymphocytes. J. Exp. Med. 134, 242. Vitetta E. S. & Uhr J. W. (1973) Synthesis. transport, dynamics, and fate of cell surface Ig and alloantigens in murine lymphocytes. Transplunr. Rec. 14, 50. Vitetta E. S., Yuan D., Krolick K., Isakson P., Knapp M., Slavin S. & Strober S. (1979) Characterization ot a spontaneous murine B cell leukemia (BCL, ). III. Evidence for monoclonality using anti-idiotypic antibody. J. Immunol. 122, 1649. Williams P. B.. Kubo R. T. & Grey H. M. (1978) Ii-Chains from a nonsecretor B cell line differ from secreted Iwzhains at the C-terminal end. J. Immunol. 121, 2435. Yuan D.. Uhr J. W. & Vitetta E. S. (1980) A peptidediffcrcnce between /t chains from cell associated and secreted IgM of the BCL, tumor. J. Inznzunol. 125, 40.