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1999 Beef Program Report. The Department of Animal Sciences Colorado State University.

STUDIES ON THE NATURE OF LEPTIN BINDING PROTEINS IN SHEEP

R. S. Yemm and K. L. Hossner

SUMMARY

Leptin is a newly discovered hormone that regulates feed intake, body composition and reproduction. The current studies were designed to examine the characteristics of leptin-specific serum binding proteins in sheep. Different approaches to leptin radioiodination were examined to provide a suitable marker for these studies. A modified Chloramine T method provided the most consistent results. Storage of ¹²⁵I-leptin at 4⁰ C in buffered 0.1% Triton X-100 and 5% glycerol reduced the formation of leptin aggregates and increased the amount of native ¹²⁵I-leptin. Chromatography of sheep serum equilibrated with ¹²⁵I-leptin demonstrated leptin binding to a serum component with a molecular weight of 186,000 Daltons. These studies suggest that one or more binding proteins for leptin are present in sheep serum and provide the basis for additional studies in sheep and cattle.

KEY WORDS: leptin, sheep, serum binding

INTRODUCTION

Leptin is a recently discovered hormone that is important in the regulation of animal body composition, appetite and reproduction. Mice that lack a functional leptin gene are obese, and replacement of leptin in these animals results in a loss of appetite and a concomitant loss of body fat and normalization of body composition. In addition, leptin induces early puberty in rodents and is likely to have direct, positive effects in many organs of the reproductive system (see review by Hossner, 1998).

Leptin circulates in the bloodstream in a native form (molecular weight = 16,000 Daltons) and as a reversible, high molecular weight complex with large serum binding proteins. The binding proteins provide a reservoir of circulating leptin and are thought to regulate its biological action. Other well-studied hormones (such as steroids and IGFs) which have serum binding proteins are, in general, inactive when bound to their binding proteins. The current studies were initiated to examine the physical and biological properties of leptin serum binding proteins in sheep. These studies showed that leptin is difficult to iodinate consistently, outline some of the practical problems in working with leptin, and provide initial data about the nature of ovine serum leptin binding proteins.

METHODS AND MATERIALS

Initial attempts to radioiodinate recombinant ovine (o) leptin (kindly provided by Dr. D. Keisler, Univ. of Missouri) utilized the Iodogen reagent (Pierce Chemical). Ovine leptin (3 mg) in 50 ml of 0.1 M NaPO₄, pH 7.1, was combined with Na¹²⁵I (1 mCi) and incubated for 8 minutes at room temperature in the presence of the Iodogen reagent (1 mg). Several attempts using this method were unsuccessful at incorporating ¹²⁵I into the leptin molecule. Chloramine T was used in subsequent leptin radioiodination reactions. Ovine leptin (3 mg) in 50 ml of 0.5 M NaPO₄, pH 7.5, was combined with 1.0 mCi of Na¹²⁵I and 15 ml of Chloramine T (30 mg). The reaction was allowed to proceed for 1.5 minutes at room temperature, and was halted by the addition of 3 ml of sodium metabisulfate (6 mg). This procedure resulted in radioiodinated leptin with specific activities ranging from 8-81 mCi/mg of peptide and a high proportion of aggregated, high molecular weight leptin.

The Chloramine T reaction was modified to create milder conditions for the iodination of ovine and human (h) leptin (gift, Eli Lily). Chloramine T was repurified by extraction with carbon tetrachloride before use, the reaction was allowed to proceed for 3 minutes, and was halted by the addition of 40 ml (400 mg) of L-cysteine hydrochloride. Radioiodinated leptin was separated from the unreacted Na¹²⁵I by Sephadex G-25 chromatography in 50 mM NaPO₄, pH 7.4, containing 0.1% Triton X-100. Fractions (1 ml) were collected and counted in a gamma counter to assess ¹²⁵I incorporated into the leptin molecule. This method resulted in specific activities of 60-296 mCi/mg protein and less aggregation of the ¹²⁵I-leptin product.

Sephadex G-50 chromatography was used to assess the quality of the radioiodinated leptin preparations immediately after iodination and after storage in various reagents chosen to reduce the formation of aggregation products. A Sephadex G-50 column (1 x 48 cm) was pre-equilibrated with 50 mM NaPO₄, pH 7.4, containing 0.1% Triton X-100. One-third or one-half of the ¹²⁵I-Leptin stock was applied to the column, and the column was eluted with the equilibration buffer. Fractions (1 ml) were collected and assessed for ¹²⁵I by counting on an automatic gamma counter (Micromedic 4/200+). Several reagents were tested for their ability to stabilize the iodinated leptin during storage. Aliquots (0.1 ml) of stock ¹²⁵I-h-Leptin were stored for 7 days at 4⁰ C in either 5% glycerol, 0.1 M L-cysteine hydrochloride, or 1% ascorbic acid (all in column buffer). Sephadex G-50 chromatography was performed as described above to assess the ratio of aggregated high molecular weight species to ¹²⁵I-h-Leptin peaks.

The nature of leptin-specific binding proteins in ovine serum was examined by incubating 0.5 ml of sheep serum with 0.5 ml of 50 mM NaPO₄, pH 7.4, and 750,000 cpm of ¹²⁵I-h-Leptin for 16 hr at 4°C. The reaction mix was applied to a column (2.5 x 48 cm) of Sephacryl S-300 (Pharmacia) which was pre-equilibrated and eluted with 50 mM NaPO₄, pH 7.4. Fractions (3 ml) were collected and assessed for radioactivity by the automatic gamma counter. A serum protein elution profile was obtained by measuring absorbance at 280 nm. An aliquot of ¹²⁵I-h-Leptin was chromatographed to provide a radioactive profile of native leptin in the absence of binding proteins. Molecular weight standards were chromatographed and measured at 280 nm to provide a standard curve to determine the size of serum leptin binding proteins. The standards used and their molecular weights were: carbonic anhydrase (29,000), bovine serum albumin (66,000), alcohol dehydrogenase (150,000) and apoferritin (443,000).

Studies were performed to determine optimum conditions for a competitive protein binding assay for the leptin binding proteins. The assay was performed in 50 mM NaPO₄, pH 7.4, with 3.13, 6.25, and 12.5 ml of ovine serum in 500 ml reaction volume containing 10,000 cpm of ¹²⁵I-h-Leptin. The buffer components tested to optimize specific binding included BLOTTO (5% Carnation Non-fat Dry Milk), BLOTTO plus 0.1% Triton X-100, 0.1% Triton X-100, and 1% bovine serum albumin (BSA). Samples were incubated for 16 hr at 4°C. After incubation, 100 ml rabbit g-globulin (15 mg/ml in assay buffer) was added to each tube and vortexed, followed by the addition of 16% polyethylene glycol (1 ml). Tubes were again vortexed and incubated on ice for 20 minutes. Assay tubes were then centrifuged for 30 minutes at 1700 x g, and supernatant liquids were aspirated. The pellets were counted in an automatic gamma counter, and specific binding was defined as: (Total Binding cpm – Non-specific cpm) / Total cpm. Non-specific binding was defined as the radioactivity which was precipitated immediately after the addition of ¹²⁵I-leptin to the reaction tubes (zero-time nonspecific binding).

RESULTS AND APPLICATION

Radioiodination of leptin has proven to be problematic for many investigators. Leptin is susceptible to damage by both strong oxidizing and reducing agents. Most iodination protocols use a strong oxidizing reagent (such as Chloramine T) to introduce radioactive iodine into the tyrosine residues of proteins. The oxidation reaction is stopped by the addition a strong reducing agent, such as sodium metabisulfate, followed by separation of the unreacted reagents and free radioiodine from the iodinated protein. We have previously used a relatively mild iodination protocol with success with a variety of proteins. Unfortunately, this protocol, using the Iodogen reagent, was not effective with leptin. When a relatively mild Chloromine T procedure was used, iodine was successfully incorporated into leptin, but most of the product consisted of a high molecular weight aggregate. This aggregate elutes from Sephadex G50 in the void volume, with a molecular weight of at least 50,000.

We employed two measures to reduce oxidation/reduction damage: Chloramine T was extracted with carbon tetrachloride to reduce the amount of highly reactive dichloro-Chloramine T and cysteine was used as mild reducing agent to stop the iodination reaction. In addition, the nonionic detergent Triton X-100 was added to column and storage buffers to reduce leptin aggregation during purification and storage. While these modifications alleviated some immediate aggregation problems, we saw that the amount of aggregated ¹²⁵I-leptin increased with time of storage. As shown in Figure 1, ¹²⁵I-leptin stored for 7 days in 0.1% Triton X-100/phosphate buffer (control) eluted from the G50 column primarily as a high molecular weight aggregate (fraction 14), with a trailing shoulder of native leptin. When ¹²⁵I-leptin was stored in 1% ascorbate, an antioxidant, only the aggregate form of ¹²⁵I-leptin was present. Storage in cysteine resulted in a low molecular weight form of ¹²⁵I-leptin (fractions 27-29), intermediate in size between authentic leptin (fraction 18) and Na¹²⁵I (fraction 35). We found that the addition of 5% glycerol to the storage buffer reduced leptin aggregation and we were able to recover 50-60% of the ¹²⁵I-leptin for use in binding studies.

Figure 2 shows the calibration curve of the Sephacryl S-300 column used to study the binding of ¹²⁵I-leptin to ovine serum. The molecular weight standards eluted in log linear manner between 29,000 and 443,000 Daltons. When ¹²⁵I-leptin was chromatographed over this column (Figure 3), it eluted at fraction 36, consistent with a 16,000 Dalton protein. After overnight equilibration of ¹²⁵I-leptin with ovine serum, two radioactive peaks eluted from the column. The first peak (fraction 27) had a calculated molecular weight of 186,000 and the second peak coeluted with native leptin. The absorbance profile of ovine serum and the elution volumes of the molecular weight standards are also shown for reference in Figure 3.

Table 1 shows the effects of different protein and detergent combinations on the specific (S) and nonspecific (N) binding of ¹²⁵I-leptin to increasing concentrations (0.625, 1.25 and 2.5%) of ovine serum. While buffer supplemented with BLOTTO or Triton X-100 had relatively low and invariant levels of nonspecific binding (2-4%), specific binding was absent whenever Triton X-100 was present. When BLOTTO was the protein component of the binding buffer, dose-dependent binding was abolished. A dose-dependent increase in specific ¹²⁵I-leptin binding was observed only when 1% BSA was added to the binding buffer. In 1% BSA, nonspecific binding was relatively high, but constant over the doses of serum used.

In summary, the current report describes the difficulties in preparation and storage of radioiodinated leptin. Aggregation of ¹²⁵I-leptin occurs during the radioiodination reaction and upon storage under standard conditions. Addition of glycerol (5%) and the detergent Triton X-100 (0.1%) to the leptin buffers improved the stability of non-aggregated 125I-leptin as determined by Sephadex G50 chromatography. Initial results from Sephacryl S300 chromatography and competitive protein binding studies with 125I-leptin have provided evidence for the presence of leptin binding proteins in sheep serum. Future studies will include Western Blot analysis and competitive ligand binding studies to further characterize leptin binding proteins in sheep and cattle serum.

REFERENCE

Hossner, K. L. 1998. Cellular, molecular and physiological aspects of leptin: Potential application in animal production. Canad. J. Anim. Sci. 78(4):463-472.