Indian Journal of Experimental Biology Vol. 40, April 2002, pp. 456-466

Studies on chemical modification of ovine luteinizing hormone and its subunits with different heterobifunctional cross-linking agents

Ranjit C Singh, Vinod Singh* & Anis Alam

Hormone Biochemistry Laboratory, Institute of Self-Organising Systems and B iophysics, and Department of Biochemistry (AA), North-Eastern Hill University, Permanent Campus, Shillong 793 022, India

Received 15 December 2001

Increasing use of heterobifunctional cross-l inking agents (HBCLA) in the design of defined conjugates for selective targeting and inducing i mmune response and to understand the significance of chemical modification of -NH2 group of ly­sine residues of native oLH and its a- and �- subunits of ovine lutein izi ng hormone (oLH) on, immunological, biological ac­tivity of the hormone and a:� subunit recombination, the £-NH2 group(s) of oLH and its a- and �- subunits were separately and sequentially modified with different heterobifunctional cross-linking agents (HBCLA) such as N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), succin imidyl 6-[3-(2-pyridyldithio)propionamido) hexanoate (LC-SPDP), 2-iminothiolane (2IT), and 4-succinimidyloxcarbonyl-a-methyl-a(2-pyridyldithio)toluene (SMPT). The aoLH modified by HBCLA recombine to native �oLH was judged by reverse phase high performance chromatography (RP-HPLC) analysis. Similarly �oLH modified by HBCLA also recombine to native aoLH. The sequential modification of subunits led to pro­gressive reduction i n immunoreactivity, receptor binding and stero idogenic activity of the dimer. The modification of six or more £-NH2 groups in aoLH although recombine fully with native �oLH but fai led to show receptor binding, anti-oLH anti­body reactivity and steroidogenic activity. The modification upto four groups in aoLH compromised immunological and biological activities but further addition of two or more groups completely abolished immunological and biological activity of the recombinants indicating the importance of later two amino groups in the receptor binding and steroidogenic activity.

Pituitary glycoprotein hormones, LH, FSH, TSH and placental human and equine chorionic gonadotropin (hCG and eCG) consists of common a-subunit, which is highly conserved among the species and a hormone specific �-subunitI .2. Within a species, the a-subunit is common and can be interchanged with the hormone specific �-subunit to produce a recombinant with

*For correspondence: Tel: +9 1 -364-550-028 Fax: +91 -364-550-076 E-mail : vinodsingh56@yahoo.co. in Abbreviations: BSA, Bovine Serum Albumin; DTT, Dithiothrei­tol; eCG, Equine chorionic gonadotropin ; EDTA, Ethylene dia­mine tetra aceticacid; LHRH, Lute inizing hormone releasing hor­mone; IODOGEN, 1 ,3,4,6 tetrachloro 3a, 6a-diphenylglycouril ; oLH, Ovine luteinizing hormone; LH, Lute inizing hormone; FSH, Foll icle stimulating hormone; hCG, Human chorionic gonadotro­pin; RP-HPLC, Reverse-phase high performance l iquid chroma­tography; RPMI, Roswell Park Memorial Institute; RIA, Radio­i mmunoassay; RRA, Radioreceptor assay; SPDP, N-Succinimidyl 3-(2-pyridyldithio)propionate; LC-SPDP, Succin imidyl 6-[3-(2-pyridyldithio)propionamido) hexanoate and SMPT, 4-succinimidyloxcarbonyl-a-methyl-a(2-pyridyldithio)toluene; TFA, Trifluroacetic acid; HBCLA, Heterobifunctional cross-l inking agent; hFSH, Human foll icle stimulating hormone; DTNB, Ell­man's reagent; EDTA, Ethylene diamine tetra acetic acid; 2-IT, 2-Iminothiolane.

specificity dictated by the choice of �- subunit l .2 . Sev­eral investigations have been carried out to understand the significance of different amino acid side chains in both subunits to determine the effect on immunoreac­tivity and biological activity of the recombinants (for re�iews see 3-5). Generally, most of such chemical modifications on the a-subunit cause a drastic loss in the recombination ability to �-subunit and therefore loss in receptor binding as well as biological activity. The �-subunit is less sensitive to such chemical modi­fications comparative to a-subunie-5. The chemical modification of E-NH2 group(s) of oLH with HBCLA have been carried out6•7• All these reagents abolish positive charge of lysine after modification and sig­nificantly affected the immunoreactivity and biologi­cal activity of the dimeric hormone8-I O• The present communication describes a systematic study on the modification of E-NH2 group of a- and �- subunits of oLH with HBCLA its effect on subunit-subunit re­combination, receptor binding, immunoreactivity and biological activity.

Materials and Methods Ovine LH used in the present investigation was pu­

rified from the lyophilized sheep pituitary powder and

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SINGH et at.: CHEMICAL MODIFICATION OF OVINE LUTEINIZING HORMONE 457

the subunits were prepared by following the published procedure 1 1 . 1 2. The RP-HPLC analysis was performed to assess the purity of u- and /3- subunits. lodogen TM,

21T, SPDP, LC-SPDP, SMPT, Ellman's reagent and DTT were obtained from Pierce Chemicals Co. (Rockford, IL, USA). Sephadex G-25 and G- I00 were obtained from Pharmacia fine Chemicals Co., USA. BSA (Cohn fraction IV) was purchased from Sigma Chemicals, St. Louis, MO, USA. All other chemicals used were of highest purity available.

Modification of &-NH2 groups with HBCLA The HBCLA modification(s) of oLH was carried

out following essentially the procedure described ear­lier6• 13. The subunits were reacted with HBCLA in the different molar ratios in the initial reaction as shown in Table 1 . The reaction mixture incubated at room temperature for 30 min. was passed through Sephadex G-25 ( 1 .5 x 30 cm) column. The column was devel­oped with using 50 mM ammonium bicarbonate. The protein peak appearing in the void volume (Vo) was pooled, lyophilized and stored at 4°C. The extent of modification was determined as described earlier6• 13 .

(a) Modification of &-NH2 groups with SPDP/ LC­SPDP

The SPDP/LC-SPDP modificatiion of oLH was carried out as described earlier6• 1 3• The oLH was re­acted with SPDP/LC-SPDP in the molar ratios vary­ing from 1 :0.5, 1 : 1 , 1 :2, 1 :3, 1 :4, 1 :5, 1 :6, 1 : 8, 1 : 1 0, 1 :20 and 1 :30 (mole/mole) in the initial reaction. In a typical reaction, the hormone dissolved in sodium phosphate buffer ( 1 00 mM, pH, 7.5 containing 150mM NaCI) was activated with SPDP/LC-SPDP at various concentrations. The SPDP/LC-SPDP was dis­solved in a minimal volume of dry absolute ethanol and added slowly to the oLH subunit solution with gentle stirring. The reaction was carried out at room temperature for 30 min and passed through Sephadex G-25 ( 1 .5 x 30 cm) column, using 50mM ammonium bicarbonate. Fractions of 1 .5ml were collected and read at 280nm. The protein peak appearing in the void volume (Vo) was pooled, lyophilized and stored at 4°C. The extent of modification was determined on the basis of liberation of pyridine-2 thione obtained upon DTT treatment l 3. From the absorption at 1.343 nm which was monitored in a UV -VISIBLE spectropho­tometer, the concentration of the liberated pyridine-2-thione was calculated by using extinction coefficient

3 of pyridine-2-thione, 1.343 = 8 .08xlO M"I . The absorp-

tion at 280nm was corrected for pyridine-2-thione contribution l3 .

(b) Modification of &-NH2 groups with 2fT The amino groups of u- and /3- subunits and oLH

were modified was carried out by following the pro­cedure described earlier'4- '6 . Briefly, the oLH subunits were dissolved in 50mM triethanolamine-HCl con­taining 100 mM NaCl, ImM EDTA and the pH was adjusted to 8.0 with 0.2M NaH2P04• The oLH was reacted with 21T in the molar ratios varying from 1 :0.5, 1 : 1 , 1 :2, 1 : 3, 1 :4, 1 :5, 1 :6, 1 : 8 and 1 : 10 (mole/mole) in the initial reaction. The reaction was terminated by adding 100 III of borated buffer-saline containing 2.2 M glycine. The derivatized oLH was then treated for 1 hr at room temperature with 2 mM DTNB, the Ellman's reagent l? The mixture is passed through Sephadex G-25 column equilibrated with ni­trogen flushed buffer consisting of 100mM sodium phosphate buffer (PH 7 .4), 1 00mM NaCI and ImM EDTA. The number of activated disulfide groups in­troduced into the oLH was determined by reducing the DTNB treated sample with DTT and measuring the absorption of released 3-carboxylate 4-nitrothio­phenolate ion which has molar absorptivity of 1 .36 x 1 04 M-1 cm- I at 4 12 nm17•

(c) Modification of &-NH2 groups with SMPT The SMPT modification of u- and /3-subunit of

oLH was carried out following the procedure de­scribed earlier? Briefly, The subunits dissolved in 50mM borate buffer, pH 9.0 containing 1 .7% NaCI were reacted with SMPT (dissolved in DMF) in the molar ratios varying from 1 :0.5, 1 : , 1 , 1 :2, 1 :4, 1 :6, 1 : 8 , 1 : 10 and 1 :20 (mole/mole) in the initial reaction. In a typical reaction, the subunit dissolved in 50 mM borate buffer, pH 9.0 containing 1 .7% NaCI were re­acted with SMPT (dissolved in DMF) was activated with SMPT at various concentrations. The reaction was carried out at room temperature for 30 min. and a 5-fold molar excess of DTT dissolved in the borate buffer was added. To this reaction mixture, a solution of 5,5' -dithio-bis(2-nitrobenzoic acid) [Ellman's re­agent] in the dry DMF was added and the mixture was stirred gently for 60 min. at room temperature and was passed through Sephadex G-25 ( 1 .5x30 cm) col­umn, equilibrated in nitrogen-flushed phosphate­EDT A buffer. The fraction of 1 .5 ml were collected and read at 280nm. The protein peak appearing in the void volume (V 0) was pooled and concentrated in an

458 INDIAN J EXP BIOL, APRIL 2002

Amicon ultrafiltration cell fitted with YM2 mem­brane. The extent of modification was determined on the basis of liberation of 3-carboxylato-4-nitrothio­phenolate ion obtained upon DTT treatment which has a molar absorptivity of 1 .36x104 M-1 cm-I at 4 1 2 nm17•

Recombination studies The recombination experiments were performed in

50 mM sodium phosphate buffer (PH 7.2, containing 100 mM NaCI). Briefly, equal amount of aoLH, aoLH modified with 2IT/SPDP/ LC-SPDP/SMPT were mixed with the native f30LH subunit and incu­bated at room temperature for 24-72 hr. Similar re­combination protocol was used for native f3oLH, f3oLH-2IT/SPDPILC-SPDP/SMPT derivatives. The recombination mixture was fractionated on Sephadex G- 100 column. The aoLH, aoLH-2IT, aoLH­SPDPILC-SPDP/ or SMPT and their recombinants with native f30LH or f3oLH-2IT, f3oLH-SPDPILC­SPDP/ or SMPT and their recombinants with native aoLH were subjected to RP-HPLC analysis using a continuous gradient as described earlier6-9.

Radioiodination of oLH The highly purified oLH was radioiodinated by 10-

dogen™ methodl 8 as described for LHRHI9. The spe­cific activity of the radioiodinated e251-oLH) ranged from 40-60 IlCilllg. The 1 251_oLH was stored at -20°C and used within 4 weeks of preparation for RIA and RRA.

Immunoreactivity determination The immunoreactivity was determined by competi­

tive displacement method as described earlier6-9. All samples and other reagents were diluted in RIA buffer (50 mM sodium phosphate buffer, pH 7.5 containing 1 50 mM NaCI and 0. 1 % each BSA and sodium azide). Appropriately diluted anti-oLH antibody (giv­ing 30-50% of binding in the absence of unlabelled hormone) was mixed with the competing hormone in the RIA system. The bound and free 1251_oLH was separated by the addition of 0.20 ml of 1 :5 diluted Pansorbin™ (Calbiochem, CA). The supernatant con­taining unbound 1251_oLH was removed by aspiration and the pellet was counted in a LKB-mini gamma counter (-70% efficiency).

Receptor binding activity determination The receptor binding activity was determined in a

radioreceptor assay (RRA) by using rat testicular

homogenate as a source of receptor6-9. Briefly, the radioiodinated oLH (0.5- 1 ng; - 1 ,60,000- 1 ,80,000 CPM/tube), the test samples and RRA buffer (25 mM Tris-HCI, pH 7.5, containing I OmM magnesium chlo­ride and I mg/ml BSA) were incubated at 20°C for 1 6-20 hr. After incubation, the reaction was terminated by addition of 2ml RRA buffer and centrifuged in cold at 3,000 g for 20 min. The supernatant was re­moved by aspiration and pellet was counted as de­scribed above.

Steroidogenic activity determination The progesterone induction ability of oLH and the

recombinants was determined with ley dig tumor cells, MLTC- l which possess receptors for gonadotropins2o. Upon binding of gonadotropins to MLTC- l cell, the progesterone is secreted into the medium. The tumor cells were cultured in a 24-well plates in RPMI- 1640 media supplemented with 1 0% FCS and antibiotics as described20. After culturing -50,000 cells/well for 48 hr, the wells were washed thrice times with serum free medium and incubated with different concentra­tions of test samples. The incubation was carried out in a humidified chamber with 95% oxygen and 5%

. carbon dioxide. An aliquot was removed for proges­terone estimation using specific RIA as described earlierlO.

RP-HPLC analysis RP-HPLC analysis of native oLH, oLH subunits,

HBCLA modified subunits and their recombinants were carried out by using a stepwise gradient of H20 and CH3CN having 0. 1 % TFA as described earlier6-9.

Results

Modification of oLH and a- and fJ subunits The isolated subunits of oLH were subjected to RP­

HPLC analysis to further establish purity of the sub­units before modification and recombination studies. The HPLC purified subunits were used in subsequent HBCLA modifications and recombination studies. Table 1 shows the initial molar ratio used in the modi­fication and E-NH2 modified. The initial molar ratio of 1 :0.5, 1 : 1 , 1 :2, 1 :3 , 1 :4, 1 :5 , 1 :6, 1 :8 and 1 : 10 (mole/mole) yielded 1 :0.4, 1 :0.6, 1 :0.8, 1 : 1 , 1 :2 1 :2.4, 1 :4 and 1 :6 respectively (in aoLH-2IT), 1 :0.5, 1 :0.7, 1 : 1 .5 , 1 :2, 1 :2.2, 1 :3.2, 1 :4, 1 :5 .6 and 1 :7.2 respec­tively (in aoLH-SPDP) and 1 :0.5, 1 :0.7, 1 : 1 .3 , 1 : 1 .6, 1 : 1 .8, 1 :3 . 1 , 1 :3.2, 1 :4.6 and 1 :5 .6 respectively (in

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SINGH el al.: CHEMICAL MODIFICATION OF OVINE LUTEINIZING HORMONE 459

aoLH-LC-SPDP) (Table 1 ) . Less number of -NH2 group modifications with LC-SPDP comparative to SPDP may also attribute to the increase in the carbon chain length in LC-SPDP. Fig. 1 shows the chemical structures of oLH, aoLH and �oLH modified deriva­tives used in the present communication. The number of amino group(s) modified are recorded in a Table 1 . The degree of modification of amino groups in aoLH was relatively higher than that of the native oLH therefore indicating that the -NH2 groups in the iso­lated subunits are more reactive than the native oLH.

Three different molar ratio of �oLH:2ITILC-SPDPI or SPDP ( 1 : 1 , 1 :3 and 1 :5 ) were used which resulted into all the £-NH2 groups modification. Higher molar ratio was not used as the introduction of more LC­SPDP or SPDP groups induced hydrophobicity in the molecule resulting into the precipitation of the pro­tein.

Recombination studies In order to determine whether 21T or SPDP or LC­

SPDP or SMPT modified oLH subunits recombine with the respective native aoLH or �oLH, the recom­bination studies were carried out. The recombination mixture was fractionated on a precalibrated G- l00 column. The protein eluting before native subunit was collected and further analysed.

RP-HPLC analysis The HBCLA modified subunits and their recombi­

nant products were subjected to RP-HPLC analysis to determine the extent and the site of -NH2 modifica­tions. The RP-HPLC chromatograms of aoLH shows two major peaks, the peak at 4.78 and t7.36. A progres­sive modification occurring in aoLH which is evident from the emergence of several new peaks at t7.6 and few shoulders at t8.4 and t9.8 . However, a gradual shift of t7.6- 15. 1 was observed in different molar ratio modi­fications6,7. 16. The shift of t7.36 towards the higher re­tention in time may be due to introduction of hydro­phobic groups to the suBunits6.7, 16. Under the similar conditions, the thiolation of native oLH exhibited lit­tle different RP-HPLC chromatograms6. It is therefore difficult to state whether the £-NH2 groups of native oLH involved in thiolation are the same as in the thiolated aoLH. Similar to aoLH, �oLH when se­quentially modified shows the different RP-HPLC chromatograms of �oLH and its modified derivatives. The �oLH eluted as a duplet at t 14. 1 and t I 5. 14 . A 1 : 1 modification resulted into the appearance of many

peaks at t14.6, t 1 5.2 and t 16.0 A 1 :3 resulted into t I4.9. t 15.4, and t I 5. 16' Further modification of �oLH ( 1 :5) intro­duced the hydrophobicity and the retention time was further shifted7, 16.2 1 -23.

To further confirm that the modified subunits in­deed recombine with the respective subunit, RP­HPLC was carried out on the top tube of the purified recombinant mixture on a Sephadex G- 1 00 column. The gel-filtration chromatography on Sephadex G-1 00 and RP-HPLC chromatograms of recombinants of native aoLH and �oLH with different modified oLH subunits show a recombination derivative. The RP-HPLC analysis clearly demonstrate the presence of native �oLH duplet at t 14.20 and t 1 5. 10 in the hybrid­ized derivative . Similarly, the other �oLH- modified derivatives also hybridized to native aoLH subunit and eluted at t and t. 7. 16.2 1 -23 . 7.4 '4.7

Immunoreactivity, receptor binding and steroidogenic activity

In order to study the immunological and biological properties of native oLH, subunit recombinants, anti­oLH antibody reactivity, receptor binding and steroi­dogenic activities were determined. The immunoreac­tivity was assessed by using anti-oLH antibodies in a competitive displacement assay and receptor recogni­tion in radioreceptor assay using rat testicular ho­mogenate 6-9. The quantitative immunoreactivity data are recorded in Table 2. Subsequent modifications with LC-SPDP and SPDP showed progressive inhibi­tion in the reactivity (Table 2). Similar observations were also made for the receptor binding property of the recombinants (Table 3). Both immunoreactivity and receptor binding activities were drastically inhib­ited even after a minor modification (Tables 2, 3). The receptor binding and immunoreactivity were com­promised upto 2-3 -NH2 modifications but subsequent modifications although retained some immunoreactiv­ity but the receptor binding activity was drastically reduced (up to 0. 1 %). Contrary to aoLH the �oLH-2IT/LC-SPDP/SPDP modified derivatives upto two -NH2 groups retained substantially both immunoreac­tivity and receptor binding (Table 3) thereby showing that the -NH2 groups of lysine in the aoLH are more crucial for receptor binding than that of -NH2 groups of lysine in the �oLH. The steroidogenic activity as determined by the rat testicular homogenate and 1251_

oLH as tracer was found to be unaffected in the low molar ratio modified derivatives (up to 1 :3) in the aoLH-2IT:�oLH recombinants). Higher modification

460 INDIAN J EXP BIOL, APRIL 2002

Table 1 - Relationship between the moles of SPDP, LC-SPD and SMPT added and the number of amino groups modified in a and /3 subunits

Sample Code Molar ratio used No. of -NHl groups Molar ratio obtained (Subunit:2ITILC-SPDP/SPDP modified (Subunit: 2ITILC-SPDP/SPDP

or SMPT) or SMPT)

aoLH modification

a oLH-2IT-0.5 1 :0.5 0.4 1 :0.4 a oLH-LC-SPDP-0.5 1 :0.5 0.5 1 :0.5 aoLH-SPDP-0.5 1 :0.5 0.5 1 :0.5 aoLH -SMPT -0.5 1 :0.5 0.5 1 :0.5 aoLH-2IT- I .0 1 : 1 0.6 1 :0.6 aoLH-LC-SPDP-1 I : 1 0.7 1 :0.7 aoLH-SPDP- I .O 1 . 1 0.7 1 :0.7 aoLH-SMPT- 1 .0 1 : 1 0.5 1 :0.5 aoLH-2IT-2.0 1 :2 0.8 1 :0.8 aoLH-LC-SPDP-2 1 :2 1 .3 1 : 1 .3 aoLH-SPDP-2 1 :2 1 .5 1 : 1 .5 aoLH- SMPT-2.0 1 :2 0.8 1 :0.8 aoLH-2IT-3 1 :3 1 .0 1 : 1 .0 aoLH-LC-SPDP-3 1 :3 1 .6 1 : 1 .6 aoLH-SPDP-3 1 :3 2.0 1 :2.0 aoLH- SMPT-3.0 aoLH-2IT-4 1 :4 1 .0 1 : 1 .0 aoLH-LC-SPDP-4 1 :4 1 .8 1 : 1 .8 (aoLH-SPDP-4 1 :4 2.2 1 :2.2 aoLH- SMPT -4.0 1 :4 1 .5 I : 1 .5 aoLH-2IT-5 1 :5 2.0 1 :2.0 aoLH-LC-SPDP-5 1 :5 3. 1 1 :3 . 1 aoLH-SPDP-5 1 :5 3.2 1 :3.2 aoLH-2IT-6 1 :6 2.4 1 :2.4 aoLH-LC-SPDP-6 1 :6 3.2 1 :3.2 aoLH-SPDP-6 1 :6 4.0 1 :4.0 aoLH- SMPT-6.0 1 :6 2.8 1 :2.8 aoLH-2IT-8 1 :8 4.0 1 :4.0 aoLH-LC-SPDP-8 1 :8 4.6 1 :4.6 aoLH-SPDP-8 1 :8 5.6 1 :5.6 aoLH- SMPT-8.0 1 : 8 3.2 1 :3.2 aoLH-2IT- 1 O 1 : 10 6.0 1 :6.0 aoLH-LC-SPDP- I O 1 : 10 5.6 1 :5.6 aoLH-SPDP- 10 1 : 10 7.2 1 :7.2 aoLH- SMPT- I O.0 1 : 10 3.6 1 :3.6

jX>LH modification /3oLH-21T- 1 1 : 1 0.4 1 ;0.4 /3oLH-LC-SPDP- 1 1 : 1 0.4 1 :0.4 /3oLH-SPDP- I 1 : 1 0.5 1 :0.5 /3oLH-SMPT - 1 .0 1 : 1 0.4 1 :0.4 /3oLH-2IT-3 1 :3 1 .0 1 : 1 .0 /3oLH-LC-SPDP-3 1 :3 1 .2 1 : 1 .2 /3oLH-SPDP-3 1 :3 1 .3 1 : 1 .3 /3oLH- SMPT-3.0 1 :3 1 .0 1 : 1 .0 /3oLH-2IT-5 1 :5 1 .4 1 : 1 .4

/3oLH-LC-SPDP-5 1 :5 2.0 1 :2.0

/3oLH-SPDP-5 1 :5 2 . 1 1 :2 . 1

/3oLH- SMPT-6.0 1 :6 1 .8 1 : 1 .8

"The numbers 0.5- 10 indicates the use of various ratios of oLH:2IT, SPDP, LC-SPDP or SMPTin the reaction. Three sepa­rate experiments indicated 5- 1 0% variation in the extent of modification. "Three different experiments showed 5-8% devia­tion in immunoreactivity and 5 - 1 2% in the receptor binding activity.

...

SINGH et al.: CHEMICAL MODIFICATION OF OVINE LUTEINIZING HORMONE 46 1

oLfUaoLfU�LfUSPDP

oLfUaoLfU�LHVLC-SPDP protein-�-�-CH -CH -CH -S -�' 2 2 2

N -

oLfUaoLfU�LHI2IT (Blocked with Ellman's reagent)

� �H+a � protein-N-C-CI-i2 C�- CHrS-v v- N�

COO'

H

oLfUaoLfU�LfUSMPT (Hindered -S-S- bond) protein-�-��-s-� . v"=!_ I �\NJ-

CH2

Fig. I -Chemical structure of oLH, aoLH and �LH subunits modified with the use of different cross-linking agents, 2IT, SMPT, LC-SPDP and SPDP.

affected more on steroidogenic activity of the aoLH-2IT:(30LH. Interestingly, 1 : 1 modification enhanced the steroidogenic activity. This may be due to the in­troduction of hydrophobic groups in the aoLH which might have affected in the inhibition of receptor rec­ognition but enhanced the steroidogenic activity (Table 3). No appropriate explanation could be given regarding this observation. The retention of biological activity may be due to the fact that only 1 % receptor occupancy is required to induce and sustain full bio­logical activity.

Discussion The increasing use of heterobifunctional cross­

linking agents in synthesis of hormone-carrier conju­gate

.s for �elec�ively targeting to the �cific cells8. 10.

24.

29

or mducmg Immune response30-3 prompted us to study the effect of NH2 group modification by HBCLA in the isolated subunits on their recombina­tion, immunoreactivity, receptor binding and biolo§i­cal activity. Ovine LH contains 12 E-NH2 groupSI . 7. Ten of which are in the aoLH and two in the (3oLH. Three types of chemical modification studies were carried oue7-39. The introduction of acidic group (such as maleyl) or neutral group (like alkyl or carbamyl) or basic (such as guanidination or acetimidination), it became clear that the most critical amino acids are in aoLH37.39. The lack of E-NH2 in the 13 subunit in the pig and rat further justifies the importance of -NH2

groups in the a-subunit4 . The carbamylated or maley­lated intact oLH was inactive37. Moreover the carba­mylated or maleylated oLH subunits recombined de­rivatives were also completely inactive37 . The acyla­tion of E-NH2 groups could modify only 1 0 amino groups. Possibly, the acylating agent could not reach to these 2 unreactive amino groups as these amino groups were buried towards the site of subunit-subunit interaction37. The positive charge of amino groups of oLH and its subunit play an important role in the sub­unit recombination, receptor binding and biological activity. Interestingly, the positive charge on the (3oLH is not crucial for subunit-subunit interaction37.39. Therefore, more attention is given to aoLH than 130LH subunit.

The alpha subunit consists of 88-97 residues and there are five highly conserved disulfide bonds40.41 . Out of these three comprise the cystein knot40.41 . It is ob­served that the Asn56 oligosaccharide is the most im­portant oligosaccharide for coupling receptor binding to signal transduction, whereas the Asn82 oligosaccha­ride is important for subunit folding40.41 . The alpha oLH has three variable and two conserved regions. The first variable region (sequence 1 -8) is not important as this sequence is frequently missing. The second and third variable region consisting of residues 1 5-33 and 68-85 respectively correspond to the hairpin loops L1 and L34.40.4 1 . The conserved region of long-loop L2 and the C-terminus possess putative receptor binding

462 INDIAN J EXP BIOL, APRIL 2002

regions as defined by peptide-walking experiments of The crystal structure of HF-treated hCG indicates the hCG and hFSH and both are involved in subunit asso- presence of only limited helicity in the ex helix region ciation. It is believed that because the alpha subunit (residues 40-46) being conserved is important for the must combine with as many as four different beta sub- biological activity40.4 1.42. The exoLH sequence Pr044-units, subunit association regions are highly conserved. Ala45-Arg46-Ser47-Lys48-Thr50 is homologous to the

Table 2-Immunoreactivity, receptor binding and steroidogenic activity of oLH, oLH modified with 2-iminothiolane, SPDP and LC-SPDP

[The activities are expressed as percentage with respect to native oLH which was taken as 100%. The method of calculating % activity is described in Material and Methods. EDso is the dose causing 50% displacement)

oLHlmodified oLH" No. of -NH2 Immunoreactivitl Rece�tor bindingb Steroidogenicb modified EDso % Cross-reactivity EDso % Cross-reactivity activity (%)

oLH 0.75 100.00 0.98 100.00 100 oLH* oLH-IT-O.5 0.40 0.90 83.33 3.20 30.60 100- 1 10 oLH-SP-0.5 0.50 3.40 22.05 4.90 20.00 20-80 oLH-LCSP-0.5 0.50 3.75 20.00 4.40 22.20 60-80

oLH-IT- 1 .0 0.60 0.80 93.75 3.50 28.00 100- 1 10 oLH-SP- 1 .0 0.70 3.40 22.05 5.90 16.6 1 ND oLH-LCSP- 1 .0 0.70 6.25 12.00 20.40 4.80 ND

oLH-IT-2.0 0.80 1 .30 57.69 5.40 18 . 14 60-90 oLH-SP-2.0 1 .50 4.68 1 6.02 2 1 .00 4.66 20-70 oLH-LCSP-2.0 1 .30 8.33 9.00 49.00 2.00 10-30

oLH-IT-3.0 1 .00 2.30 32.60 6.30 1 5.55 70- 1 10 oLH-SP-3.0 2.00 5.30 14. 1 5 32.00 3.06 30-80 oLH-LCSP-3.0 1 .60 1 0.50 7 . 14 6 1 .00 1 .60 10-30

oLH-IT-4.0 1 . 10 2.65 28.30 7.80 1 2.56 60-80 oLH-SP-4.0 2.20 6.20 1 2.09 32.60 3.00 20-40 oLH-LCSP-4.0 1 .80 1 2.50 6.00 54.40 1 .80 10-40

oLH-IT-5.0 1 .20 2.50 30.00 7.50 1 3.06 50-80 oLH-SP-5.0 3.20 7.50 10.00 49.00 2.00 10-30 oLH-LCSP-5.0 3 . 10 1 8.70 4.01 98.00 1 .00 8- 10

oLH-IT-6.0 2.40 3.60 20.80 15 . 10 6.49 40-70 oLH-SP-6.0 4.00 8.70 8.62 8 1 .50 1 .20 8-20 oLH-LCSP-6.0 3.20 22.00 3.40 1 22.00 0.80 2-6

oLH-IT-8.0 4.00 6.20 1 2.09 49.00 2.00 8-20 oLH-SP-8.0 5.60 12.00 6.25 245.00 0.40 2-6 oLH-LCSP-8.0 4.60 375.00 0.20

oLH-IT- 1O 6.00 7.50 10.00 8 1 .70 1 . 19 2- 10 oLH-SP- 1O 7.20 16.30 4.60 490.00 0.20 oLH-LCSP- 1O 5.60

oLH-IT-20 7.00 1 2.50 6.00 196.00 0.50 2-6 oLH-SP-20 9.00 46.80 1 .60 oLH-LCSP-20 6.00

oLH-IT-30 9.00 37.50 2.00 980.00 0. 10 2-4 oLH-SP-30 9.00 oLH-LCSP-30 6.00

"The numbers 0.5-30 means the use of various ratios of oLH:2IT, SPDP or LC-SPDP in the reaction. Three separate experi­ments indicated 5- 10% variation in the extent of modification. �hree different experiments showed 5-8% deviation in immuno­reactivity , 5- 1 2% in the receptor binding and 10-20% in steroidogenic activity. oLH-2IT, oLH-SPDP, oLH-LC-SPDP modifi­cation data were obtained from ref. 16.

�

SINGH et al.: CHEMICAL MODIFICATION OF OVINE LUTEINIZING HORMONE 463

Table 3-Immunoreactiv ity and receptor binding properties of different recombinants of IT, LC-SPOPI SPOP or SMPT modified subunits with respective native subunits

Steroidogenic Sample Code' Immunoreactivitl Receetor Bindingb activity

EOso % Activity EOso % Activity (% )

oLH 0.80 100 0.96 100 100 aoLH. �LH 0.62 100 0.80 100 100 oLH* 0.90 100 1 . 10 100 100 aoLH. fk>LH* 0.70 100 0.90 100 100

f}oLH recombinants

aoLH-2IT-0.5.fk>LH 1 .02 78.0 (60.78) 2.40 40.0 (33.33) 90- 1 10 aoLH-LC-SPOP-0.5f3oLH 2.28 35.0 (27.20) 2.52 38.0 (31 .74) 1 10- 1 30 aoLH-SPOP-0.5. f30LH 2.66 30.0 (23.30) 5.85 16.4 (1 3.67) 1 10- 1 30 aoLH-SMPT-O.5: f3oLH* 1 .60 56.25(43.75) 2.75 40.00« 32.72) 1 10- 1 30 aoLH-2IT- If3.oLH 0.99 80.2 (62.62) 2.63 36.4 (30.4 1 ) 90- 1 1 5 aoLH-LC-SPOP- I .f3oLH 2.85 28.0 (21 .75) 5.05 19.0 (1 5.84) 1 10- 130 aoLH-SPOP- I . �LH 3.33 24.0 ( 1 8.61 ) 7.60 12.6 ( 10.52) 1 10- 1 30 aoLH-SMPT-1 .0: fk>LH* 2. 10 42.80(33.33) 3.90 28.20(23.07) 1 10-120 aoLH-2IT-2f3.oLH 1 .33 60.0 (46.6 1) 4.48 2 1 .4 ( 17.85) 80-100 aoLH-LC-SPOP-2.f3oLH 4.84 1 6.5 ( 1 2.80) 16.0 6.0 (5.00) 100- 1 10 aoLH-SPOP-2. f30LH 4.44 1 8.0 ( 1 3.96) 9.60 10.0 (8.33) 100- 1 10 aoLH-SMPT-2.0: fk>LH* 2.50 36.00(28.00) 6.90 1 5.94( 13.04) 90- 1 10 aOLH-2IT-3.f3oLH 1 .3 1 44.0 (34.25) 5.33 1 8.0 ( 1 5.00) 70-90 aoLH-LC-SPOP-3.�LH 6.66 12.0 (9.30) 48.0 2.0 ( 1 .66) 90- 1 1 0 aoLH-SPOP-3. fk>LH 4.44 1 8.0 ( 1 3.96) 12.0 8.0 (6.66) 100- 1 10 aoLH-2IT-4.fk>LH 2.42 33.0 (25.61 ) 6.23 15 .4 ( 12.84) 60-80 aoLH-LC-SPOP-4.f3oLH 8.42 9.5 (8.42) 96.0 1 .0 (0.83) NO aoLH-SPOP-4. �LH 7.61 10.5 (8. 14) 16.27 5.9 (4.90) NO aoLH-SMPT -4.0: f3oLH* 4.30 20.93( 16.28) 1 8.30 6.01 (4.90) 10-40 aoLH-2IT-5.fk>LH 2.81 28.4 (22.06) 7.74 12.4 (20.33) 60-80 aoLH-LC-SPOP-5.f3oLH 13 .8 5.8 (4.50) 1 92.0 0.5 (0.41 ) 4- 10 aoLH-SPOP-5. fk>LH 9.09 8.8 (6.82) 34.28 2.8 (2.33) 4-6 aoLH-SMPT-6.0: fk>LH* 9.00 10.00(7.77) 36.60 3.00(2.45) 8-10 aoLH-2IT-6.�LH 3.57 22.4 ( 17.36) 1 1 .70 8.2 (6.83) 40-60 aoLH-LC-SPOP-6.fk>LH 32.0 2.5 ( 1 .94) NO aoLH-SPOP-6. fk>LH 16.0 5.0 (3.87) 96.00 1 .0 (0.83) 2-6 aoLH-SMPT-8.0: f3oLH* 36.00 2.50(1 .94) 1 3.75 0.08(0.06) 2-6 aoLH-2IT-8.fk>LH 4.93 16.2 ( 12.57) 1 5.73 6. 10 (5.25) 10-20 aoLH-LC-SPOP-8.fk>LH NO aoLH-SPOP-8. fk>LH 50.00 1 .6 ( 1 .24) 1200.0 0.08 (0.06) 1 -6 aoLH-SMPT- IO.0: fk>LH* 1 80.0 0.50(0.39) aoLH-2IT- IO.f3oLH 10.0 8.0(6.20) 40.0 2.40(2.00) 4- 10

' . . aoLH-LC-SPOP- IO.fk>LH NO aoLH-SPOP-IO.0. fk>LH NO aoLH-SMPT-20.0 : fk>LH*

aoLH recombinants

�LH-IT - l .aoLH 0.90 88.0 (68.80) 1 .65 58.0 (58.48) 90- 1 10 f3oLH-LC-SPOP- I .aoLH 2.85 28.0 (21 .74) 4.36 22.0 ( 1 8.34) 1 10- 130 �LH-SPOP-I .aoLH 3.08 26.0 (20. 12) 5.33 1 8.0 ( 15.00) 1 10-130 fk>LH-SMPT-l .aoLH* 1 .80 50.00(38.88) 2.40 45.83(37.50) 1 10- 140 �LH-IT-3.aoLH 1 .02 78.2 (60.78) 1 .84 52.0 (43.47) 90- 100 �LH-LC-SPOP-3.aoLH 4.44 1 8.0 ( 1 3.96) 6.40 1 5.0 ( 12.50) 80- 1 10 fk>LH-SPOP-3.aoLH 5.33 1 5.0 ( 1 1 .63) 9.60 10.0 (8.33) 80- 1 10 fk>LH-SMPT-3.aoLH* 2.30 39. 10(30.43) 5.20 2 1 . 1 5( 17.30) 90- 1 1 0 �LH-IT-5.aoLH 1 .48 54.0 (41 .89) 2.28 42.0 (35.08) 70- 100 �LH-LC-SPOP-5.aoLH 80.00 1 .0 (0.77) 16.00 6.0 (5.00) 30-60 fk>LH-SPOP-5 .. aoLH 1 6.00 5.0 (3.87) 27.40 3.5 (2.91 ) 20-40 fk>LH-SMPT-5 .. aoLH* 9.30 9.67 (7.52 10.00 1 1 .00 (9.00) 26-45

Number in parentheses were obtained when (oLH.(oLH was taken as lOO%.aThe numbers 0.5- 10 indicates the use of vari-ous ratios of oLH:2IT, SPOP, LC-SPOP or SMPT in the reaction. bThree different experiments showed 5-8% deviation in immunoreactivity, 8-12% in the receptor binding activity and 10-20% in steroidogenic activity. NO, not determined. *These data were obtained from ref. 7. Other data were obtained from ref. 1 6.

464 INDIAN J EXP BIOL, APRIL 2002

a helices sequence 40-46 of hCG40. It has been dem­onstrated that the aoLH can specifically be nicked by endoproteinase at the Arg46-Ser4743 . The S-S of nicked aoLH broken by sulfitolysis generated N- and C- terminal 1 -44 and 47-96 fragments respectivel/3 . Interestingly, these could be reoxidized to give a re­constituted nicked aoLH that was capable of reasso­ciation with BoLH43. This holoprotein showed only 2-3% receptor binding. The first and the second amino group modification with LC-SPDP showed 6-2% re­ceptor binding (Table 2). Possibly, the second modifi­cation was in close proximity to this helical region and the helicity was influenced by these modification.

As proposed, the region 44-50 of aoLH is in the close proximity to the BoLH determinant loop and found to be important in hormone receptor interac­tion4.44• Using site directed mutagenesis by replacing Lys44 with Ala in ahCG (corresponding Lys48 in aoLH) did not appreciably alter the association with the BoLH but the receptor binding was abolished45• Thus, there is strong evidence from endoproteinase cleavage, site directed mutagenesis and X-ray crystal­lography that this region of the a-subunit is important in the receptor binding.

Presently described chemical modification studies differ with that of the studies carried out earlier' ·4 . In all earlier reports, all the available amino groups were modified and its effect on biological activity was de­termined whereas we sequentially modified the -NH2 groups and its effect on immunological and biological activity was determined. The modification of more than 4 -NH2 groups in the isolated aoLH although recombine fully with the native BoLH but did not show oLH immunoreactivity, receptor binding and steroidogenic activity (Table 2). The modification of 2-3 -NH2 groups of aoLH with 2IT compromised immunological and biological activities but further introduction of hydrophobic LC-SPDP groups in the aoLH abolished both the immunological and biologi­cal activities (Table 2). Therefore, indicating the im­portance of the later two -NH2 groups in the receptor recognition and steroidogenic activity. As described, the actual sites of the critical NH2 groups of the posi­tions 49, 55, 57, and 95 might be involved in receptor recognition46. Like SPDP, the introduction of more hydrophobic LC-SPDP group in the B-subunit did not alter the quaternary structure of the hormone as no dissociation of the subunit takes place. The compari­son of immunoreactivity and receptor binding proper-

ties of 2IT modified oLH with SPDP or LC-SPDP modified derivatives of oLH clearly reveal that the introduction of LC-SPDP into oLH affected more drastically as immunbreactivity and receptor binding activities were further inhibited (Table 2). However, the steroidogenic activity was relatively less affected (Table 2). We have no rational explanation at the pre­sent time for this puzzling observation but others in previous studies have also noted higher potency for some derivatives of oLH in which E-NH2 groups of lysine were modified47. It is possible that the increase in the hydrophobicity might have induced an orienta­tion of the hormone-receptor complex in the cells, which produce higher progesterone production in the cells. In some studies, alkyl substitution in the NH2 groups of oLH, the adenylate cyclase activation and hormone-receptor interaction have been reported48. Catt and Duffau49 studied the minimum requirement of receptor occupancy to induce biological response. Interestingly, only 1 % receptor occupancy is required for full steroidogenic response.

Acknowledgement This investigation was supported by a grant from

UGC, New Delhi as a UGC-Career Award to VS.

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