Multiple forms of lactadherin (breast antigen BA46) and butyrophilin are secreted into human milk as major components of milk fat globule membrane

Abstract

In the lactating mammary gland, epithelial cells secrete triacylglycerols in the form of droplets enveloped by an apical surface membrane. This membrane is known as the milk fat globule membrane (MFGM; Mather & Keenan, 1983). MFGM-associated proteins have been studied and employed in developing antibodies against surface antigens of breast epithelial cells, which are used in breast cancer immunodiagnosis and histopathology (Salinas et al. 1987; Larocca et al. 1991; Peterson et al. 1995). So far, only a small number of proteins have been analysed and their sequence identified in bovine, murine and human MFGM; among them are butyrophilin (Jack & Mather, 1990; Ishii et al. 1995; Taylor et al. 1996), MFG-E8 (Stubbs et al. 1990), PAS 6/7 (Hvarregaard et al. 1996) and lactadherin or breast antigen BA46 (Couto et al. 1996; Taylor et al. 1997). Several minor proteins have yet to be characterized, since it is not easy to isolate them in large quantities from the membrane. SDS gel patterns give useful information about MFGM proteins, such as concentration, relative molecular mass and presence of carbohydrate. Over forty membrane components have been separated by electrophoretic techniques from bovine MFGM (Mather et al. 1980).The research reported here combined SDS-PAGE with sequencing analysis and describes the composition of human MFGM, with the exception of high molecular mass mucin, which only penetrates an acrylamide gel of 40 g/l. Mucins have been extensively studied and the sequence predicted from cDNA (Gendler et al. 1990). Surprisingly, identification of the protein bands in the present study revealed that three proteins alone constituted the major components of human MFGM: xanthine oxidase (EC 1.1.3.22), butyrophilin and lactadherin. Lactadherin belongs to a family of proteins possessing epidermal growth factor-like and factor V/VIII C1/C2-like domains, including bovine PAS 6/7, guinea pig GP55 (Hvarregaard et al. 1996) and murine MFG-E8 (Stubbs et al. 1990). In a previous investigation, we characterized lactadherin (formerly breast antigen BA46) and its truncated 30 kDa form as components of healthy human MFGM (Giuffrida et al. 1998). Human butyrophilin has recently been cloned and sequenced (Taylor et al. 1996); the presence of two extracellular immunoglobulin superfamily domains suggested a potential cell surface receptor function. This study was aimed at identifying and characterizing the multiple forms of the major proteins of MFGM.

Human proteome enhancement: High-recovery method and improved two-dimensional map of colostral fat globule membrane proteins

“…The glycophosphoform of b-casein is probably a precursor for many peptides that occur in milk and are generated in the mammary gland by proteolysis. a-Lactalbumin (spot 23) was identified in a previous study [16] confirming the hypothesis of Patton and Huston [47] of a membrane-associated version of the soluble protein. Further investigation of possible post-translational modifications is necessary to understand the partition of this molecule between different milk fractions.…”

“…Proteins from SDS-PAGE were electroblotted onto a PVDF membrane (Problott; Applied Biosystems, Foster City, CA, USA) in a semi-dry transfer cell (Bio-Rad) as described elsewhere [16], with the only difference that 10% instead of 20% methanol was used. Proteins were visualized by staining with 0.1% Coomassie Brilliant Blue R-250 in 40% methanol, 1% acetic acid for 1 min and destaining with 50% methanol.…”

Analysis of major proteins and fat fractions associated with mare's milk fat globules

“…1), with M r ranging from 50 to 55 kDa, are probably glycosylation variants of a peripheral membrane glycoprotein called lactadherin. Lactadherin was also identified in spots 11 and 12 at lower M r (42 kDa), probably being a truncated form of these glycoforms [30]. Lactadherin is a multidomain protein: human lactadherin has an epidermal growth factor domain in the N-terminal part of the sequence, while in other species, such as the cow, mouse and pig, there are two similar domains [16].…”

Proteolysis of Milk Fat Globule Membrane Proteins in Preterm Milk: A Transient Phenomenon with a Possible Biological Role?

“…In general, MFGM proteins are multidomain glycosylated proteins; some truncated forms have already been identified, but their full-length forms were always present in the same samples (25)(26)(27). Lactadherin consists of two N-terminal EGFlike domains followed by two repeated domains, homologous with the C I and C2 domains of blood clotting factors V and VIII (28); medin, an internal cleavage product (from residue 245 to residue 294) of lactadherin, has been found in aortic amyloid deposits (29) and internal elastic lamina of the temporal artery (30).…”