Identification of Histidine-Rich Glycoprotein in Human Colostrum and Milk

Abstract

ABSTRACT. Histidine-rich glycoprotein (HRG) is a 74-kD glycoprotein, originally discovered in plasma, which contains an unusually large amount of histidine (13 mol%) and proline (13 mol%). The specific functions of this protein remain unclear, although it binds (reversibly) transition metal ions such as Cu(I1) and Zn(1I) with high capacity (10-13 equivalent) and moderate to high affinity (h = 0.2-10 pM). Because the bioavailability of Cu (I1) and Zn(I1) ions in human milk is high, we have used specific antibodies from polyclonal antisera directed against purified human plasma HRG to investigate whether this or a related protein is a component of human colostrum and(or) mature milk. Fresh human colostrum (d 1-3) and milk (d 4-120) were collected in the presence and absence of multiple protease inhibitors and EDTA. Immuno "dot" blot analyses and ELISA were developed; HRG was present in both colostrum (0.13-10 pg/mL) and mature milk (0.1-10 pg/mL). Unidentified components in colostrum and milk, however, were found to depress HRG antigenicity in these assays. Western transfer and immunoblots of denatured colostrum and milk samples analyzed by SDS-PAGE revealed the presence of an immunoreactive band at 74-78 kD, with other bands at 47 and 24 kD under both reducing and nonreducing conditions; smaller immunoreactive fragments (12-14 kD) were detected in some samples. We observed at least one additional band of immunoreactivity of greater molecular mass (>I10 kD) in colostrum under nonreducing conditions; we did not observe these bands in plasma samples. Immunoaffinity and Zn(I1) affinity isolation of HRG from colostrum and milk resulted in the copurification of several associated proteins. Nterminal amino acid sequence analysis of the isolated 74-kD immunoreactive protein provided further evidence for identification as intact HRG; 13 of the first 15 residues were identical. These results demonstrate the presence of HRG, or a structurally related protein, in human colostrum and milk. Understanding the biology and function of trace elements in human. milk must include the identification of specific human milk peptides and proteins that influence metal ion transport (bioavailability) and storage in the mammary gland during lactogenesis, as well as in the gastrointestinal tract of breast-fed infants. Equally important, in our view, is the need to understand the role of transition metal ions in the regulation of milk protein structure, bioavailability, and function. Much can be learned from existing knowledge of the transport and use of transition metal ions by serum proteins. For example, in several species, including humans, many of the proteins in maternal serum, particularly the metal-binding proteins, are similar or identical to the whey proteins in colostrum (1,2).Adult human serum contains a protein discovered in 1972 (3) referred to as histidine-rich glycoprotein.' HRG is a 74-kD glycoprotein that contains significant quantities of both histidine (13%) and proline (13%) (4), which are concentrated primarily in the C...

Presence in Human Skeletal Muscle of an AMP Deaminase-associated Protein That Reacts with an Antibody to Human Plasma Histidine-Proline-rich Glycoprotein

“…It was originally isolated from human plasma (Heimburger et al 1972) but its presence has been demonstrated also in platelets and milk (Leung et al 1984;Hutchens et al 1992). Its specific function remains unclear, although several biological roles have been suggested.…”

The Role of Histidine-Proline-Rich Glycoprotein as Zinc Chaperone for Skeletal Muscle AMP Deaminase

“…Plasma is the major pool of HPRG, but it is also found in infant urine, colostrums, milk, platelets, megakaryocytes and immuno cells such as monocytes and macrophages [58,63,64]. The tissue distribution of mouse HPRG mRNA, investigated by Northern blot analysis performed on total RNA from liver, spleen, thymus, heart, lung, kidney, brain and testis, demonstrated that HPRG mRNA is produced only in the liver [65].…”

New Insights into the Functions of Histidine-Rich Glycoprotein

“…Plasma is the major pool of HRG, but it is also found in infant urine, colostrums, and milk (Hutchens et al, 1992;Yip and Hutchens, 1991). The concentration of HRG in plasma is 100-150 mg/L (Heimburger et al, 1972;Lijnen et al, 1981b;Morgan et al, 1978).…”