Characterization of human epidermal growth factor in human serum and urine under native conditions

Abstract

The objective of this study was to investigate the molecular nature of the human epidermal growth factor (EGF) in serum and urine samples of normal subjects. Recombinant EGF emerged as a single peak and did not interact with human IgG1 and albumin up to the concentration of 12 μg/ml. Freshly separated human serum contained only trace amounts of EGF. However, EGF appeared and increased in serum separated from blood after spontaneous overnight clotting. The authentic 6 kDa form of EGF made up nearly 40% of the total EGF in serum and revealed relatively homogeneous feature. The remaining immunoreactive fractions corresponded to 160 kDa proEGF. Immunoreactive EGF in blood seemed to be associated with the EGF release from platelets. TSKgel G3000SW chromatography of freshly-voided morning and day urines revealed that urine samples mainly contained two major form of EGF; a high-molecular-weight (HMW) and low-molecular-weight (LMW) forms. In the sense of molecular nature of EGF contents, morning urine was more heterogeneous than day urine of the same individuals. The LMW form of EGF in morning urine, in which its proportion was more than 90% of the total EGF, revealed further heterogeneous feature generally containing three to four different components.

Introduction

Epidermal growth factor (EGF) is a globular protein of nearly 6 kDa consisting of 53-amino acids [1]. Epidermal growth factor is originally discovered in the mouse submaxillary gland [2] and has been subsequently shown to stimulate the proliferation and differentiation of epidermal and epithelial cells and serves as a potent mitogen for a variety of cell types [3], [4]. Human EGF is synthesized as a very long prepro-protein of 1168–1207 amino acids from which the factor itself (i.e. mature EGF, position 970–1023) is released by proteolytic cleavage [5], [6], [7]. This large molecule contains a hydrophobic domain allowing the precursor to be anchored in the plasma membrane [7]. However, the mechanism by which prepro-EGF is processed to mature EGF is not well understood. It was proposed that in cells incapable of cleaving this precursor, such as kidney cells, the membrane-bound form itself might also serve as a receptor for as yet unknown ligands [7], [8]. Although epidermal growth factor is found in varying concentrations in milk, saliva, urine, and serum [9], [10], there are conflicting reports in the literature regarding the exact nature of EGF molecules (i.e. proEGF or monomeric mature EGF) under native conditions [7], [8], [11], [12], [13], [14], [15].

Numerous evidences indicated that EGF and its receptor might play an important role in different clinical situations including carcinogenesis, chronic obstructive pulmonary disease, neonatal necrotizing enterocolitis, and skin inflammation and the high-molecular-weight form of EGF seemed to be related with the expression of the transformed phenotype [16], [17], [18], [19], [20]. Therefore, discriminative analysis of EGF, in the sense of molecular-weight form under native conditions, seemed to be important for investigating the relationship of EGF with certain types of pathological conditions.

The relative amounts of high- and low-molecular-weight forms of EGF in urine have been debated, since different results have been obtained by different techniques. In order to address the problem it is important to quantitate the different molecular forms of EGF with a well-established method or combined methods.

The purpose of this study is to develop a processing-independent ELISA that is capable of quantitating different molecular forms of human EGF with equimolar potency and analyze proEGF and mature EGF in fractionated human serum and urine samples using a well-established size-exclusion chromatography technique.