Changes in Lactoferrin and Lysozyme Levels in Human Milk During the First Twelve Weeks of Lactation

The structure of human lactoferrin has been refined crystallographically at 2.8 A (1 A = 0.1 nm) resolution using restrained least squares methods. The starting model was derived from a 3.2 A map phased by multiple isomorphous replacement with solvent flattening. Rebuilding during refinement made extensive use of these experimental phases, in combination with phases calculated from the partial model. The present model, which includes 681 of the 691 amino acid residues, two Fe3+, and two CO3(2-), gives an R factor of 0.206 for 17,266 observed reflections between 10 and 2.8 A resolution, with a root-mean-square deviation from standard bond lengths of 0.03 A. As a result of the refinement, two single-residue insertions and one 13-residue deletion have been made in the amino acid sequence, and details of the secondary structure and tertiary interactions have been clarified. The two lobes of the molecule, representing the N-terminal and C-terminal halves, have very similar folding, with a root-mean-square deviation, after superposition, of 1.32 A for 285 out of 330 C alpha atoms; the only major differences being in surface loops. Each lobe is subdivided into two dissimilar alpha/beta domains, one based on a six-stranded mixed beta-sheet, the other on a five-stranded mixed beta-sheet, with the iron site in the interdomain cleft. The two iron sites appear identical at the present resolution. Each iron atom is coordinated to four protein ligands, 2 Tyr, 1 Asp, 1 His, and the specific Co3(2-), which appears to bind to iron in a bidentate mode. The anion occupies a pocket between the iron and two positively charged groups on the protein, an arginine side-chain and the N terminus of helix 5, and may serve to neutralize this positive charge prior to iron binding. A large internal cavity, beyond the Arg side-chain, may account for the binding of larger anions as substitutes for CO3(2-). Residues on the other side of the iron site, near the interdomain crossover strands could provide secondary anion binding sites, and may explain the greater acid-stability of iron binding by lactoferrin, compared with serum transferrin. Interdomain and interlobe interactions, the roles of charged side-chains, heavy-atom binding sites, and the construction of the metal site in relation to the binding of different metals are also discussed.

The effects of the duration of lactation upon lactoferrin, lysozyme, total IgA, SIgA, SIgA antibodies to Escherichia coli somatic antigens and leukocytes in human milk were investigated. Longitudinal and cross-sectional studies were performed with milk collected from women 20 to 35 years of age during te first year of lactation. Collection and storage conditions and immunologic analyses were controlled to minimize confounding variables. The concentrations of lactoferrin, total IgA, and leukocytes and the uptake of 3H-thymidine by phytohemagglutinin-stimulated lymphocytes fell during the first several weeks of lactation; afterward, the levels of lactoferrin and IgA stabilized. Approximately 90% of total IgA in human milk during the year was SIgA. Secretory IgA antibody titers to E. coli increased in some individuals studied longitudinally suggesting that the enteromammary gland pathway of SIgA antibody production was active after several weeks of lactation. Moreover, the concentrations of lysozyme, after falling to a nadir of 20 to 30 micrograms/ml at 2 to 4 weeks, rose to 200 to 300 micrograms/ml by six months and remained elevated. The immunologic system in human milk undergoes remarkable changes which may represent adaptations for the recipient infant.