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Identification of Lactoferrin Complexes in Bovine Mammary Secretions During Mammary Gland Involution1
Identification of Lactoferrin Complexes in Bovine Mammary Secretions During Mammary Gland Involution1 H. WANG and W. L. HURLEY2 Department of Animal Sciences, University of Illinois, Urbana 61801
ABSTRACT Part of the antimicrobial activity of lactoferrin resides in its ability to bind to bacteria. The complexing of lactoferrin with other proteins could alter its activity. This study identified the presence of lactoferrin complexes in mammary secretions during mammary gland involution and determined the proportion of free and complexed lactoferrin in mammary secretions. Mammary secretions were collected from Holstein cows on d 7, 14, and 21 of involution. Proteins were fractionated from defatted, filtered mammary secretions by sucrose density gradient ultracentrifugation and by gel filtration chromatography. Proteins contained in separated fractions were identified by SDS-PAGE. The presence of lactoferrin was confirmed by immunoblot analysis. Lactoferrin was present as complexed forms of high molecular mass in mammary secretions at each day of involution. The majority of lactoferrin was present in complexes of higher molecular mass rather than as monomers. A majority of lactoferrin existed in fractions of approximately 250 kDa, although peaks of lactoferrin at 150, 300, and 800 kDa were also found. The presence of lactoferrin complexes may result from interactions with casein or immunoglobulins or from the formation of lactoferrin multimers in the secretions. The interaction of lactoferrin with other proteins in mammary secretions during involution may affect the antimicrobial properties of lactoferrin. ( Key words: lactoferrin, milk proteins, mammary gland, involution) Abbreviation key: Lf = lactoferrin, MM = molecular mass, Sepharose CL-48 = sepharose crosslinked with 4% beaded agarose. INTRODUCTION In the dairy cow, mammary gland involution is initiated by cessation of milk removal. The process of
Received October 10, 1997. Accepted March 13, 1998. 1Supported by Illinois Agricultural Experiment Station as part of Hatch Projects 35-0315 and 35-0354. 2To whom correspondence should be addressed. 1998 J Dairy Sci 81:1896–1903
involution marks the functional and structural transition of the gland from a lactating state to a nonlactating state (11). During involution, secretions in the gland undergo extensive compositional changes. Components specific to milk, including lactose, fat, casein, b-LG, and a-LA, decline in content during early involution. In contrast, other components such as leukocytes, hydrolytic enzymes, Ig, serum albumin, and lactoferrin ( Lf) , increase in concentration during involution. The concentration of Lf in the gland increases considerably during active involution and becomes a major component of mammary secretions (11, 23, 25). Lactoferrin is thought to be important as part of the host defense mechanisms of mammals (25). A variety of functions have been ascribed to Lf, including antimicrobial properties, regulation of myelopoiesis, the control of hyposideremia during systemic inflammation, inhibition of antibody synthesis, enhancement of hydroxyl radical production by neutrophils, regulation of leukocyte cytotoxic activity, and lymphocyte proliferation (3, 16, 18, 24). Although iron binding is a common feature in many Lf functions, it is not required for all functions. Also, Lf commonly binds to macromolecules, such as Ig, casein, secretory component, albumin, lysozyme, and b-LG (12, 15), as well as to DNA and heparin (29). Evidence for complexes of Lf and Ig comes from efforts to isolate either of these proteins from mammary secretions (22, 28), and complexed Lf-IgG2 has been identified ( 4 ) . In addition, self-association of Lf has been suggested by the presence of Lf of high molecular mass ( MM) in bovine milk during experimentally induced infection of a mammary gland ( 9 ) . The mean MM of the Lf peak was consistent with a trimer at the peak of the infection, although the size decreased as the infection subsided. The biochemical nature and the functional significance of complexes between Lf and other macromolecules are unknown. One mechanism by which Lf exerts its antimicrobial actions involves direct binding to bacteria (6, 12). Consequently, Lf bound to other macromolecules might reduce the efficacy of the antimicrobial actions of the protein. Alternatively, Lf complexed with other antimicrobial proteins, such as Ig, might result in enhancement of antimicrobial ac-
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tivity. Such effects on the antimicrobial nature of secretions in the involuting mammary gland may be important with regard to the susceptibility of the gland to IMI. Incidence of mastitis is particularly high in dairy cows during early involution, but the gland becomes more resistant to infection in the mid dry period (19). The relatively low antimicrobial activity of mammary secretions during early involution is thought to arise from the relatively low Lf concentrations and the competition for iron binding with citrate (25), but may also result from the physical form of Lf in the mammary secretions during the involution period. Studies that biochemically and functionally characterize Lf complexes in mammary secretions may provide insight into the role of this protective protein in the mammary gland. The objectives of this study were to determine whether Lf complexes exist in mammary secretions during involution and to determine the relative amount of complexed and free or monomeric Lf. MATERIALS AND METHODS Materials Bovine Lf (for chromatography) was supplied by DMV International (Fraser, NY). Bovine Lf (purified antigen for ELISA) was from ICN Pharmaceuticals Inc. (Costa Mesa, CA). Goat anti-rabbit IgG (blotting grade) conjugated with alkaline phosphatase was from Bio-Rad Laboratories (Richmond, CA). Nitrocellulose membrane, BSA, sepharose crosslinked with 4% beaded agarose ( Sepharose CL-4B) , and gel filtration MM standards were from Sigma Chemical Co. (St. Louis, MO). Sucrose (ultrapure) and prestained protein MM standards were from Life Technologies (Gaithersburg, MD). Metricel membrane filter was purchased from Gelman Sciences, Inc. (Ann Arbor, MI). Ultra-clear™ (14 × 89 mm) tubes were from Beckman Instruments Inc. (Palo Alto, CA). Sample Collection and Preparation Mammary gland secretions (20 to 50 ml) were collected aseptically from a total of 36 nonlactating Holstein cows on d 7 ( n = 14), 14 ( n = 11), or 21 ( n = 11) after cessation of milk removal. Milk was collected from one Holstein cow in midlactation for comparison of protein profiles of the dry cows. Individual quarters were screened for bacterial contamination according to the procedure described by the National Mastitis Council (17). Samples were used only from quarters that contained no bacterial growth. Skim
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secretions were prepared by centrifugation at 10,000 × g for 20 min at 4°C to remove fat and cellular debris. The supernatant was filtered through a series of filters from 25 to 0.45 mm. Filtered secretions were aliquoted and stored at –20°C until analysis. Samples from cows were used individually for sucrose density gradient centrifugation and for some gel filtration analyses; samples were pooled ( n = 8 ) by day of involution for most gel filtration analyses. Gel Electrophoresis and Immunoblotting Proteins in mammary secretions, in fractions obtained from sucrose density gradient centrifugation, and in fractions obtained from gel filtration chromatography were separated by SDS-PAGE (14). Equal volumes of each fraction were loaded per lane. Gels (12.5%) were stained with Coomassie brilliant blue. Immunoblotting was performed as described previously (27). Immunoblots were developed using primary antibody of rabbit anti-bovine Lf as described previously (22). Secondary antibody was goat antirabbit IgG conjugated with alkaline phosphatase. The protein concentration of each sample was estimated using a dye-binding assay (Bio-Rad); BSA was the standard. Sucrose Density Gradient Centrifugation Continuous linear sucrose gradients of 5 to 25% (wt/vol) were generated by pouring 11.5 ml of 15% (wt/vol) sucrose solution containing 100 mM NaCl and 25 mM Tris (pH 7.4) into each Ultra-clear™ tube and freezing the tubes at –20°C until use. The frozen sucrose solution was thawed slowly at 22°C until the density gradient was formed. The diluted sample (500 ml containing approximately 2.4 mg of total protein) was gently layered on top of the sucrose gradient, and gradient tubes were centrifuged at 100,000 × g for 20 h at 4°C. Fractions were collected by punching a hole at the bottom of the centrifuge tube with a 16-gauge needle and collecting 0.5 ml per fraction. Gel Filtration Chromatography Sepharose CL-4B fractionates proteins or protein complexes with MM ranging from 6 to 20,000 kDa. Mammary secretions were diluted in eluent buffer [100 mM NaCl, 0.02% NaN3, and 25 mM Tris (pH 7.4)], and sucrose was added to a final concentration of 2% (wt/vol). Sepharose CL-4B was equilibrated in eluent buffer and packed into the K 26/100 column (Pharmacia LKB Biotechnology, Piscataway, NJ). Journal of Dairy Science Vol. 81, No. 7, 1998
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Proteins were eluted at a flow rate of 30 ml/h. Fractions (7.5 ml each) were collected, and absorbance at 280 nm was determined for each fraction (DU-640 spectrometer; Beckman Instruments, Inc.). Aliquots of fractions were stored at –20°C for Lf immunoassay. Lf ELISA Lactoferrin in fractions from gel filtration chromatography was assayed by ELISA as described previously (23). RESULTS Sucrose Density Gradient Centrifugation Sucrose density gradient centrifugation separates proteins by mass and density and can be used to provide evidence for Lf complexes in mammary secretions. After centrifugation, proteins or complexes with lower MM remain at the top of the linear gradient, and proteins or aggregates with higher MM migrate toward the bottom of the gradient. Isolated Lf was observed only in fractions 4 to 7, representing the lower density fractions (data not shown). When skim milk from lactating cows was separated, casein was observed in all fractions ranging from the lowest density to the highest density (Figure 1a). Caseins exist in milk primarily as aggregates of multiple caseins termed micelles. The SDS-PAGE protein profile of mammary secretions from a representative cow on d 14 of involution (Figure 1b) showed that b-LG (18 kDa) was present in fractions 1 to 4, serum albumin (68 kDa) was primarily present in fractions 3 to 6, and casein was present in all fractions. Lactoferrin was present not only in fractions 4 to 7, in which the purified Lf would be expected, but also in fractions 8 to 18, indicating MM greater than the ∼80 kDa monomeric form. Immunoglobulin, represented in the SDS-PAGE by the reduced heavy and light chain bands (Figure 1b), was present in fractions 5 to 8. The majority of Lf and Ig was present in fractions 5 to 7. Coseparation of Lf with casein was also observed in many fractions from mammary secretions of nonlactating cows. Sucrose density gradient centrifugation of mammary secretions from d 7 and 21 of involution produced qualitatively similar results (data not shown). Comparison of several mammary secretions from several cows for each day of involution suggested that the presence of Lf complexes in mammary secretions during the same stage of involution was a consistent feature of these secretions among cows (data not shown). Journal of Dairy Science Vol. 81, No. 7, 1998
Figure 1. Separation of proteins in mammary secretions by sucrose density gradient centrifugation. Proteins in fractions were separated by SDS-PAGE. Gels are of proteins in a ) milk (one representative cow) and b ) nonlactating mammary secretions from d 14 of involution (one representative cow). Lanes are original sample ( O ) and 1 to 18 [fractions 1 to 18 from sucrose density gradient, respectively (lane 1 is the lowest density; lane 18 is the highest density)]. Arrows indicate lactoferrin (Lf); serum albumin (SA); Ig, heavy chain (Ig-h); caseins (CN); Ig, light chain (Ig-l); and b-LG. Molecular mass standards (left side) are in kilodaltons.
Gel Filtration Chromatography Gel filtration chromatography separates proteins by MM; proteins with a higher MM elute from the column first. The MM of unknown proteins can be estimated based on their elution relative to a standard MM chromatogram (Figure 2a). The gel filtration profile of purified Lf indicated that the peak of eluted Lf typically occurred in fraction 52 (Figure 2b). Gel filtration chromatograms of proteins in milk and in nonlactating mammary secretions on d 7 were compared. Chromatography of milk resulted in three major peaks (Figure 2c); two major peaks were observed in the chromatogram of mammary secretions collected on d 7 of involution (Figure 2d). If Lf is not complexed, then the major fraction containing Lf should coincide primarily with fraction 52. However, if Lf is complexed with other Lf molecules or with other macromolecules, then Lf should be found in fractions representing higher MM. Proteins in fractions from gel filtration chromatography were identified by SDS-PAGE and by immunoblotting in the case of Lf. Proteins in representative fractions (indicated in Figure 3a) from chromatographed mammary secretions from d 7 of involution were separated by SDS-PAGE to demonstrate the coseparation of Lf with other proteins based on MM. The protein profile of nonlactating mammary secretions on d 7 (Figure 3b) indicated that Lf was present in fractions ranging from 33 to 53, represent-
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ing MM from 1100 to 80 kDa. This result was confirmed by immunoblotting that was specific for Lf (Figure 3c). Casein was observed in fractions 33 to
Figure 2. Gel filtration chromatogram of standard proteins and mammary secretion proteins. Chromatograms are of a ) proteins with standard molecular masses (MM), b ) isolated lactoferrin ( L f ) (peak in fraction 52; ∼80 kDa), c ) proteins in milk (one representative cow), and d ) proteins in nonlactating mammary secretions from d 7 of involution (pooled sample representing secretions from eight cows). Arrows in panel a indicate peak position of each MM standard: 1 ) blue dextran (2000 kDa), 2 ) thyroglobulin (669 kDa), 3 ) apoferritin (443 kDa), 4 ) alcohol dehydrogenase (150 kDa), and 5 ) carbonic anhydrase (29 kDa). Fifty milligrams of purified Lf, 100 mg of total milk protein, and 100 mg of total proteins of nonlactating mammary secretions were loaded. Broken vertical lines indicate fraction 52 and represent the elution fraction of the peak of purified Lf in this gel filtration system. OD = Optical density.
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51, and Ig was observed primarily in fractions 43 to 53 (Figure 3b). Similar results were obtained from chromatography of mammary secretions collected on d 14 of involution (Figure 4). In that case, the chromatogram had three major peaks (Figure 4a). The protein profile of fractions of mammary secretions from d 14 of involution indicated that Lf was present in fractions ranging from 35 to 53; MM ranged from 1000 to 80 kDa (Figure 4b). This result was confirmed by immunoblotting developed against anti-bovine Lf (Figure 4c). Chromatography of mammary secretions from d 21 of involution resulted in one major protein peak (Figure 5a). Nevertheless, Lf was observed in fractions 37 to 54; MM ranged from 800 to 80 kDa (Figure 5, b and c). Relatively little casein was present in mammary secretions collected on d 14 or d 21 (Figure 4b and 5b, respectively).
Figure 3. Analysis of proteins in nonlactating mammary secretions from d 7 of involution. a ) Gel filtration chromatogram of protein in nonlactating mammary secretions (pooled sample representing secretions from eight cows; 100 mg of total protein were chromatographed). Vertical lines on the chromatogram indicate fractions that were used for SDS-PAGE analysis. OD = Optical density. b ) SDS-PAGE of proteins in fractions collected from gel filtration chromatography. Lanes are molecular mass standards ( S ) (in kilodaltons; left side of gel), original sample ( O ) , and 22 to 63 (fractions 22 to 63, respectively). Arrows indicate lactoferrin (Lf); serum albumin (SA); Ig, heavy chain (Ig-h); caseins (CN); Ig, light chain (Ig-l); and b-LG. c ) Lactoferrin immunoblot of gel as in panel b. Intact Lf is indicated by the arrow; other bands are proteolytic hydrolysis products of Lf. Journal of Dairy Science Vol. 81, No. 7, 1998
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Of the Lf measured in gel filtration fractions of mammary secretions from d 21 of involution (Figure 6b), approximately 94% was found in fractions 41 to 51; the peak of Lf represented MM approximately 250 kDa. Significant Lf was present in fractions 46 and 49, representing MM of approximately 300 and 150 kDa, respectively. The major peak of purified Lf (80 kDa) was located primarily in fraction 52 on the gel filtration chromatogram (Figure 2b). However, peak fractions containing Lf from both d 7 and 21 of involution were observed in several fractions with higher MM, including fractions 49, 47, 46, and 38, corresponding to MM of approximately 150, 250, 300, and 800 kDa, respectively. The majority of Lf (97 and 99% of total Lf for d 7 and 21, respectively) was present in fractions
Figure 4. Analysis of proteins in nonlactating mammary secretions from d 14 of involution. a ) Gel filtration chromatogram of protein in nonlactating mammary secretions (pooled sample representing secretions from eight cows; 100 mg of total protein were chromatographed). Vertical lines on the chromatogram indicate fractions that were used for SDS-PAGE analysis. OD = Optical density. b ) SDS-PAGE of proteins in fractions collected from gel filtration chromatography. Lanes are molecular mass standards ( S ) (in kilodaltons; left side of gel), original sample ( O ) , and 22 to 64 (fractions 22 to 64, respectively). Arrows indicate lactoferrin (Lf); serum albumin (SA); Ig, heavy chain (Ig-h); caseins (CN); Ig, light chain (Ig-l), and b-LG. c ) Lactoferrin immunoblot of gel as in panel b. Intact Lf is indicated by the arrow; other bands are proteolytic hydrolysis products of Lf.
Quantificaton of Lf in Fractions The Lf content in gel filtration chromatography fractions of mammary secretions from d 7 and 21 of involution was determined by ELISA (Figure 6). Lactoferrin was distributed throughout most of the peaks with higher MM on the gel filtration chromatogram for both d 7 and 21 of involution. In mammary secretions from d 7 of involution (Figure 6a), approximately 75% of total measured Lf was found in fractions 41 to 51; the peak of Lf represented a MM of approximately 250 kDa. Additionally, the shoulder on the descending side of the major Lf peak (fraction 49) contained Lf and had a MM of approximately 150 kDa. About 22% of total Lf was found in fractions 36 to 40; the peak of Lf represented MM of approximately 800 kDa. In total, 97% of total Lf was present in MM fractions that were greater than the 80-kDa monomeric Lf form (fraction 52). Journal of Dairy Science Vol. 81, No. 7, 1998
Figure 5. Analysis of proteins in nonlactating mammary secretions from d 21 of involution. a ) Gel filtration chromatogram of protein in nonlactating mammary secretions (secretions from one of two cows at d 21 of involution that were tested by gel filtration; 100 mg of total protein were chromatographed). Vertical lines on the chromatogram indicate fractions that were used for SDS-PAGE analysis. OD = Optical density. b ) SDS-PAGE of proteins in fractions collected from gel filtration chromatography. Lanes are molecular mass standards ( S ) (in kilodaltons; left side of gel), original sample ( O ) , and 22 to 62 (fractions 22 to 62, respectively). Arrows indicate lactoferrin (Lf); serum albumin (SA); Ig, heavy chain (Ig-h); Ig, light chain (Ig-l); and b-LG. c ) Lactoferrin immunoblot of gel as in panel b. Intact Lf is indicated by the arrow; other bands are proteolytic hydrolysis products of Lf.
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Figure 6. Protein distribution and lactoferrin ( L f ) content in fractions of nonlactating mammary secretions separated by gel filtration chromatography. a ) Chromatogram of mammary secretions from d 7 of involution (pooled sample representing secretions from eight cows; 100 mg of total protein were chromatographed). b ) Chromatogram of mammary secretions from d 21 of involution (secretions from one of two cows at d 21 of involution that were tested by gel filtration; 100 mg of total protein were chromatographed). Solid lines represent optical densities ( O D ) at 280 nm; dashed lines represent Lf contents per fraction as determined by ELISA. The broken vertical line indicates fraction 52 and represents the elution fraction of the peak of purified Lf in this gel filtration system.
representing MM forms greater than monomeric Lf. In addition, 75% of Lf on d 7 and 94% of Lf on d 21 were present in fractions that also contained Ig (Figures 3b and 5b); 22% of total Lf on d 7 was present in fractions containing casein. These percentages were based on the protein profiles by SDSPAGE. DISCUSSION Results from both gel filtration chromatography and sucrose density gradient ultracentrifugation indicated that Lf exists in protein complexes with high MM in nonlactating mammary secretions during involution. These Lf complexes have MM generally greater than 80 kDa and up to 1100 kDa. Coseparation of Lf with other major proteins suggests that Lf may complex with casein, Ig, or both in nonlactating mammary secretions, resulting in the apparent shift of the MM of Lf. The relative absence of casein in mammary secretions from d 14 and 21 of involution suggests that Lf may be complexing primarily with Ig or with some other component to result in separation
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in fractions with higher MM. Quantification of Lf in fractions from gel filtration chromatography indicated that the majority of Lf was present in complexes with higher MM rather than in the monomeric form. During early involution, 97% of total Lf is present in forms with high MM. Seventy-five percent of total Lf is present in fractions that contain Ig, and much of the remainder is present in fractions that contain casein. As involution progresses, there is an increase in the proportion of Lf present in fractions with MM of approximately 250 kDa. The presence of Lf with high MM complexes in the nonlactating mammary secretions is consistent with the findings of other investigators. Isolated human Lf has a pronounced tendency to become associated with acidic proteins, such as albumin or casein, and these interactions influence its electrophoretic behavior in agarose gels (10). Lactoferrin in bovine milk has also been found to be associated with other milk proteins ( 8 ) . A gel filtration fraction with a high MM (less than 500 kDa) from nonlactating mammary secretions contained Lf and casein (20). Results from the present study indicated that most of the Lf that is present in mammary secretions during d 7 and 21 of involution separates in fractions with a MM of approximately 250 kDa. The 250-kDa fractions containing Lf also contain Ig. The presence of Lf in fractions representing 150 kDa might suggest the presence of Lf dimers, and the 300-kDa fractions might suggest the presence of Lf tetramers or aggregates of two Lf molecules plus one Ig molecule. These results are also consistent with the observation that the apparent mean MM of Lf, determined by gel filtration, increases during mastitis ( 9 ) . In that case, Lf separated at approximately the size of a trimer as the infection progressed, concurrent with increasing Lf concentrations. The nature of the binding interactions that result in the formation of Lf complexes in nonlactating mammary secretions is unknown. Ionic, disulfide, and hydrophobic interactions may all be involved in the binding of Lf with other macromolecules. The binding of Lf to a monomeric IgA affinity column can be dissociated with increasing salt concentrations (22), which suggests that the binding between Lf and IgA is an ionic interaction. Lactoferrin is a strong basic protein at a neutral pH. Casein mainly exists as casein micelles that contain phosphate groups. A possible explanation of the interaction between Lf and casein may be via an ionic interaction. In addition to ionic interactions, other forces also appear to be involved in the formation of complexes. For example, disulfide reduction will separate both the secretory Journal of Dairy Science Vol. 81, No. 7, 1998
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component and Lf from IgA preparations, suggesting that disulfide bond formation may be involved in this binding interaction (28). Lysozyme is also a strong basic protein and is positively charged at a neutral pH. Hydrophobic interactions are thought to be responsible for the formation of a Lf-lysozyme complex because it is not dissociated by ion-exchange chromatography ( 5 ) . The binding relationship between Lf and Ig may involve ionic, hydrophobic, disulfide, or multiple interactions in mammary secretions. Furthermore, human Lf can undergo polymerization in fluids containing calcium, resulting in tetramer formation, which is the predominant molecular form in human serum, tears, and breast milk ( 2 ) . A complex of Lf-IgG2 was identified in colostrum by Butler ( 4 ) and was estimated at greater than 700 kDa by gel filtration chromatography. Results from the present study indicate that the MM of Lf complexes in nonlactating mammary secretions range up to 1100 kDa. This range of Lf complexes with high MM may result from the different molar ratios of Lf to Ig or Lf to casein. The size of the Lf-IgG2 complex suggests that multiple molecules of both proteins may form the aggregate ( 4 ) . A complex of Lf and lysozyme of about 110 kDa has been observed in human milk (13). However, there is very little lysozyme in bovine mammary secretions, and such a Lf-lysozyme complex is unlikely to contribute significantly to the findings of the present study. Groves ( 7 ) found significant amounts of Lf in both the acid-precipitated casein fraction and in the whey fraction from normal milk. However, little subsequent work has been done to quantify the distribution of free Lf and complexed Lf in the mammary secretions. This scenario led to the second objective of the present study, which was to determine the proportion of free Lf and complexed Lf in mammary secretions during involution. The major proportion of total Lf exists in complexes with high MM rather than as monomeric Lf. A majority of Lf exists in fractions representing a range in MM from 150 to 300 kDa. However, in secretions from d 7 of involution, approximately 22% of total Lf is also present in a peak with the MM around 800 kDa and cochromatographed with casein. As the proteolytic activity increases during involution, casein is progressively degraded ( 1 ) . Therefore, the proportion of Lf-casein complexes would be expected to decrease by d 21 of involution compared with d 7 of involution. Lactoferrin-Ig or LfLf multimers probably are the major Lf complex forms in nonlactating mammary secretions by d 21 of involution. This large proportion of Lf complexes suggests that Lf-Ig complexes or Lf-Lf multimers may be important during involution and important in the Journal of Dairy Science Vol. 81, No. 7, 1998
resistance of the bovine mammary gland to IMI during that period. The biological relevance of Lf complexes to the functions of Lf, Ig, casein, or lysozyme remains unclear. The bacteriostatic activity of milk has been attributed primarily to the presence of Lf (18, 21, 24). Complexes of Lf with other proteins or with itself might either enhance or reduce the antibacterial activity of Lf. Little evidence is available to show that Lf complexes, such as Lf-Ig or Lf-Lf multimers, enhance the antibacterial activity in mammary secretions. However, human Lf and IgA act cooperatively with increased bacteriostatic activity (26). Immunoglobulin A alone shows some inhibition of bacterial growth, but the inhibition is considerably increased in the presence of Lf. In contrast, Lf may bind to casein micelles and form Lf-casein complexes. This interaction might prevent the interaction between Lf and lipopolysaccharide on the surface of bacteria, which is one of the Lf antibacterial mechanisms (6, 12). As a consequence, the complexing of Lf with casein might decrease the antibacterial activity of Lf. Alternatively, fractions with high MM from nonlactating mammary secretions that contained both Lf and casein were found to inhibit growth of Bacillus species (20). The nature of the Lf complexes and the intermolecular interactions of Lf with other proteins found in mammary secretions requires further characterization. CONCLUSIONS Results from gel filtration chromatography and sucrose density gradient ultracentrifugation indicate that Lf is present in complexed forms with high MM in nonlactating mammary secretions. A majority of total Lf in nonlactating mammary secretions exists in fractions with a MM of approximately 250 kDa. A smaller proportion of total Lf exists in fractions containing casein, representing a MM of approximately 800 kDa. The apparent shift in the MM of Lf from the monomeric form may be due to the presence of Lf complexes such as Lf-casein, Lf-Ig, or Lf-Lf multimers in mammary secretions. As involution progresses, the proportion of Lf as Lf-Ig complexes, and Lf-Lf multimers apparently increase. Identification of Lf in complexed forms in mammary secretions may lead to a reevaluation of the relative importance of Lf as a host defense factor in the mammary gland. ACKNOWLEDGMENTS The authors acknowledge the assistance of M. Aslam in sample collection and processing. We also appreciate the technical expertise and assistance of H. Hegarty and S. Zou in this work.
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´ nchez, 15 Lampreave, F., A. Pineiro, J. H. Brock, H. Castillo, L. Sa and M. Calvo. 1990. Interaction of bovine lactoferrin with other proteins of milk whey. Int. J. Biol. Macromol. 12:2–5. 16 Machnicki, M. 1991. Biological properties of lactotransferrin. Folia Biologica (Prague) 37:65–76. 17 National Mastitis Council. 1987. Laboratory and Field Handbook on Bovine Mastitis. W. D. Hoard & Sons, Fort Atkinson, WI. 18 Nemet, K., and I. Simonovitis. 1985. The biological role of lactoferrin. Haematologia 18:3–12. 19 Oliver, S. P., and L. M. Sordillo. 1989. Approaches to the manipulation of mammary involution. J. Dairy Sci. 72: 1647–1664. 20 Oram, J. B., and B. Reiter. 1968. Inhibition of bacteria by lactoferrin and other iron-chelating agents. Biochim. Biophys. Acta 170:351–365. 21 Reiter, B. 1983. The biological significance of lactoferrin. Int. J. Tissue React. V:87–96. 22 Rejman, J. J., H. M. Hegarty, and W. L. Hurley. 1989. Purification and characterization of bovine lactoferrin from secretions of the involuting mammary gland: identification of multiple molecular weight forms. Comp. Biochem. Physiol. 93B:929–934. 23 Rejman, J. J., W. L. Hurley, and J. Bahr. 1989. Enzyme-linked immunosorbent assays of bovine lactoferrin and a 39-kilodalton protein found in mammary secretion during involution. J. Dairy Sci. 72:555–560. 24 Sanchez, L., M. Calvo, and J. H. Brock. 1992. Biological role of lactoferrin. Arch. Dis. Child. 67:657–661. 25 Smith, K. L., and S. P. Oliver. 1981. Lactoferrin: a component of nonspecific defense of the involuting bovine mammary gland. Adv. Exp. Med. Biol. 137:535–554. 26 Stephens, S., J. M. Dolby, J. Montreuil, and G. Spik. 1980. Differences in inhibition of the growth of commensal and enteropathogenic strains of Escherichia coli by lactotransferrin and secretory immunoglobulin A isolated from human milk. Immunology 41:597–603. 27 Ventling, B. L., and W. L. Hurley. 1989. Soy protein in milk replacer identified by immunoblotting. J. Food Sci. 54:766–767. 28 Watanabe, T., H. Nagura, K. Watanab, and W. R. Brown. 1984. The binding of human milk lactoferrin to immunoglobulin A. FEBS Lett. 168:203–207. 29 Zou, S., C. E. Magura, and W. L. Hurley. 1992. Heparin-binding properties of lactoferrin and lysozyme. Comp. Biochem Physiol. 103B:889–895.
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ABSTRACT Part of the antimicrobial activity of lactoferrin resides in its ability to bind to bacteria. The complexing of lactoferrin with other proteins could alter its activity. This study identified the presence of lactoferrin complexes in mammary secretions during mammary gland involution and determined the proportion of free and complexed lactoferrin in mammary secretions. Mammary secretions were collected from Holstein cows on d 7, 14, and 21 of involution. Proteins were fractionated from defatted, filtered mammary secretions by sucrose density gradient ultracentrifugation and by gel filtration chromatography. Proteins contained in separated fractions were identified by SDS-PAGE. The presence of lactoferrin was confirmed by immunoblot analysis. Lactoferrin was present as complexed forms of high molecular mass in mammary secretions at each day of involution. The majority of lactoferrin was present in complexes of higher molecular mass rather than as monomers. A majority of lactoferrin existed in fractions of approximately 250 kDa, although peaks of lactoferrin at 150, 300, and 800 kDa were also found. The presence of lactoferrin complexes may result from interactions with casein or immunoglobulins or from the formation of lactoferrin multimers in the secretions. The interaction of lactoferrin with other proteins in mammary secretions during involution may affect the antimicrobial properties of lactoferrin. ( Key words: lactoferrin, milk proteins, mammary gland, involution) Abbreviation key: Lf = lactoferrin, MM = molecular mass, Sepharose CL-48 = sepharose crosslinked with 4% beaded agarose. INTRODUCTION In the dairy cow, mammary gland involution is initiated by cessation of milk removal. The process of
Received October 10, 1997. Accepted March 13, 1998. 1Supported by Illinois Agricultural Experiment Station as part of Hatch Projects 35-0315 and 35-0354. 2To whom correspondence should be addressed. 1998 J Dairy Sci 81:1896–1903
involution marks the functional and structural transition of the gland from a lactating state to a nonlactating state (11). During involution, secretions in the gland undergo extensive compositional changes. Components specific to milk, including lactose, fat, casein, b-LG, and a-LA, decline in content during early involution. In contrast, other components such as leukocytes, hydrolytic enzymes, Ig, serum albumin, and lactoferrin ( Lf) , increase in concentration during involution. The concentration of Lf in the gland increases considerably during active involution and becomes a major component of mammary secretions (11, 23, 25). Lactoferrin is thought to be important as part of the host defense mechanisms of mammals (25). A variety of functions have been ascribed to Lf, including antimicrobial properties, regulation of myelopoiesis, the control of hyposideremia during systemic inflammation, inhibition of antibody synthesis, enhancement of hydroxyl radical production by neutrophils, regulation of leukocyte cytotoxic activity, and lymphocyte proliferation (3, 16, 18, 24). Although iron binding is a common feature in many Lf functions, it is not required for all functions. Also, Lf commonly binds to macromolecules, such as Ig, casein, secretory component, albumin, lysozyme, and b-LG (12, 15), as well as to DNA and heparin (29). Evidence for complexes of Lf and Ig comes from efforts to isolate either of these proteins from mammary secretions (22, 28), and complexed Lf-IgG2 has been identified ( 4 ) . In addition, self-association of Lf has been suggested by the presence of Lf of high molecular mass ( MM) in bovine milk during experimentally induced infection of a mammary gland ( 9 ) . The mean MM of the Lf peak was consistent with a trimer at the peak of the infection, although the size decreased as the infection subsided. The biochemical nature and the functional significance of complexes between Lf and other macromolecules are unknown. One mechanism by which Lf exerts its antimicrobial actions involves direct binding to bacteria (6, 12). Consequently, Lf bound to other macromolecules might reduce the efficacy of the antimicrobial actions of the protein. Alternatively, Lf complexed with other antimicrobial proteins, such as Ig, might result in enhancement of antimicrobial ac-
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tivity. Such effects on the antimicrobial nature of secretions in the involuting mammary gland may be important with regard to the susceptibility of the gland to IMI. Incidence of mastitis is particularly high in dairy cows during early involution, but the gland becomes more resistant to infection in the mid dry period (19). The relatively low antimicrobial activity of mammary secretions during early involution is thought to arise from the relatively low Lf concentrations and the competition for iron binding with citrate (25), but may also result from the physical form of Lf in the mammary secretions during the involution period. Studies that biochemically and functionally characterize Lf complexes in mammary secretions may provide insight into the role of this protective protein in the mammary gland. The objectives of this study were to determine whether Lf complexes exist in mammary secretions during involution and to determine the relative amount of complexed and free or monomeric Lf. MATERIALS AND METHODS Materials Bovine Lf (for chromatography) was supplied by DMV International (Fraser, NY). Bovine Lf (purified antigen for ELISA) was from ICN Pharmaceuticals Inc. (Costa Mesa, CA). Goat anti-rabbit IgG (blotting grade) conjugated with alkaline phosphatase was from Bio-Rad Laboratories (Richmond, CA). Nitrocellulose membrane, BSA, sepharose crosslinked with 4% beaded agarose ( Sepharose CL-4B) , and gel filtration MM standards were from Sigma Chemical Co. (St. Louis, MO). Sucrose (ultrapure) and prestained protein MM standards were from Life Technologies (Gaithersburg, MD). Metricel membrane filter was purchased from Gelman Sciences, Inc. (Ann Arbor, MI). Ultra-clear™ (14 × 89 mm) tubes were from Beckman Instruments Inc. (Palo Alto, CA). Sample Collection and Preparation Mammary gland secretions (20 to 50 ml) were collected aseptically from a total of 36 nonlactating Holstein cows on d 7 ( n = 14), 14 ( n = 11), or 21 ( n = 11) after cessation of milk removal. Milk was collected from one Holstein cow in midlactation for comparison of protein profiles of the dry cows. Individual quarters were screened for bacterial contamination according to the procedure described by the National Mastitis Council (17). Samples were used only from quarters that contained no bacterial growth. Skim
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secretions were prepared by centrifugation at 10,000 × g for 20 min at 4°C to remove fat and cellular debris. The supernatant was filtered through a series of filters from 25 to 0.45 mm. Filtered secretions were aliquoted and stored at –20°C until analysis. Samples from cows were used individually for sucrose density gradient centrifugation and for some gel filtration analyses; samples were pooled ( n = 8 ) by day of involution for most gel filtration analyses. Gel Electrophoresis and Immunoblotting Proteins in mammary secretions, in fractions obtained from sucrose density gradient centrifugation, and in fractions obtained from gel filtration chromatography were separated by SDS-PAGE (14). Equal volumes of each fraction were loaded per lane. Gels (12.5%) were stained with Coomassie brilliant blue. Immunoblotting was performed as described previously (27). Immunoblots were developed using primary antibody of rabbit anti-bovine Lf as described previously (22). Secondary antibody was goat antirabbit IgG conjugated with alkaline phosphatase. The protein concentration of each sample was estimated using a dye-binding assay (Bio-Rad); BSA was the standard. Sucrose Density Gradient Centrifugation Continuous linear sucrose gradients of 5 to 25% (wt/vol) were generated by pouring 11.5 ml of 15% (wt/vol) sucrose solution containing 100 mM NaCl and 25 mM Tris (pH 7.4) into each Ultra-clear™ tube and freezing the tubes at –20°C until use. The frozen sucrose solution was thawed slowly at 22°C until the density gradient was formed. The diluted sample (500 ml containing approximately 2.4 mg of total protein) was gently layered on top of the sucrose gradient, and gradient tubes were centrifuged at 100,000 × g for 20 h at 4°C. Fractions were collected by punching a hole at the bottom of the centrifuge tube with a 16-gauge needle and collecting 0.5 ml per fraction. Gel Filtration Chromatography Sepharose CL-4B fractionates proteins or protein complexes with MM ranging from 6 to 20,000 kDa. Mammary secretions were diluted in eluent buffer [100 mM NaCl, 0.02% NaN3, and 25 mM Tris (pH 7.4)], and sucrose was added to a final concentration of 2% (wt/vol). Sepharose CL-4B was equilibrated in eluent buffer and packed into the K 26/100 column (Pharmacia LKB Biotechnology, Piscataway, NJ). Journal of Dairy Science Vol. 81, No. 7, 1998
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Proteins were eluted at a flow rate of 30 ml/h. Fractions (7.5 ml each) were collected, and absorbance at 280 nm was determined for each fraction (DU-640 spectrometer; Beckman Instruments, Inc.). Aliquots of fractions were stored at –20°C for Lf immunoassay. Lf ELISA Lactoferrin in fractions from gel filtration chromatography was assayed by ELISA as described previously (23). RESULTS Sucrose Density Gradient Centrifugation Sucrose density gradient centrifugation separates proteins by mass and density and can be used to provide evidence for Lf complexes in mammary secretions. After centrifugation, proteins or complexes with lower MM remain at the top of the linear gradient, and proteins or aggregates with higher MM migrate toward the bottom of the gradient. Isolated Lf was observed only in fractions 4 to 7, representing the lower density fractions (data not shown). When skim milk from lactating cows was separated, casein was observed in all fractions ranging from the lowest density to the highest density (Figure 1a). Caseins exist in milk primarily as aggregates of multiple caseins termed micelles. The SDS-PAGE protein profile of mammary secretions from a representative cow on d 14 of involution (Figure 1b) showed that b-LG (18 kDa) was present in fractions 1 to 4, serum albumin (68 kDa) was primarily present in fractions 3 to 6, and casein was present in all fractions. Lactoferrin was present not only in fractions 4 to 7, in which the purified Lf would be expected, but also in fractions 8 to 18, indicating MM greater than the ∼80 kDa monomeric form. Immunoglobulin, represented in the SDS-PAGE by the reduced heavy and light chain bands (Figure 1b), was present in fractions 5 to 8. The majority of Lf and Ig was present in fractions 5 to 7. Coseparation of Lf with casein was also observed in many fractions from mammary secretions of nonlactating cows. Sucrose density gradient centrifugation of mammary secretions from d 7 and 21 of involution produced qualitatively similar results (data not shown). Comparison of several mammary secretions from several cows for each day of involution suggested that the presence of Lf complexes in mammary secretions during the same stage of involution was a consistent feature of these secretions among cows (data not shown). Journal of Dairy Science Vol. 81, No. 7, 1998
Figure 1. Separation of proteins in mammary secretions by sucrose density gradient centrifugation. Proteins in fractions were separated by SDS-PAGE. Gels are of proteins in a ) milk (one representative cow) and b ) nonlactating mammary secretions from d 14 of involution (one representative cow). Lanes are original sample ( O ) and 1 to 18 [fractions 1 to 18 from sucrose density gradient, respectively (lane 1 is the lowest density; lane 18 is the highest density)]. Arrows indicate lactoferrin (Lf); serum albumin (SA); Ig, heavy chain (Ig-h); caseins (CN); Ig, light chain (Ig-l); and b-LG. Molecular mass standards (left side) are in kilodaltons.
Gel Filtration Chromatography Gel filtration chromatography separates proteins by MM; proteins with a higher MM elute from the column first. The MM of unknown proteins can be estimated based on their elution relative to a standard MM chromatogram (Figure 2a). The gel filtration profile of purified Lf indicated that the peak of eluted Lf typically occurred in fraction 52 (Figure 2b). Gel filtration chromatograms of proteins in milk and in nonlactating mammary secretions on d 7 were compared. Chromatography of milk resulted in three major peaks (Figure 2c); two major peaks were observed in the chromatogram of mammary secretions collected on d 7 of involution (Figure 2d). If Lf is not complexed, then the major fraction containing Lf should coincide primarily with fraction 52. However, if Lf is complexed with other Lf molecules or with other macromolecules, then Lf should be found in fractions representing higher MM. Proteins in fractions from gel filtration chromatography were identified by SDS-PAGE and by immunoblotting in the case of Lf. Proteins in representative fractions (indicated in Figure 3a) from chromatographed mammary secretions from d 7 of involution were separated by SDS-PAGE to demonstrate the coseparation of Lf with other proteins based on MM. The protein profile of nonlactating mammary secretions on d 7 (Figure 3b) indicated that Lf was present in fractions ranging from 33 to 53, represent-
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ing MM from 1100 to 80 kDa. This result was confirmed by immunoblotting that was specific for Lf (Figure 3c). Casein was observed in fractions 33 to
Figure 2. Gel filtration chromatogram of standard proteins and mammary secretion proteins. Chromatograms are of a ) proteins with standard molecular masses (MM), b ) isolated lactoferrin ( L f ) (peak in fraction 52; ∼80 kDa), c ) proteins in milk (one representative cow), and d ) proteins in nonlactating mammary secretions from d 7 of involution (pooled sample representing secretions from eight cows). Arrows in panel a indicate peak position of each MM standard: 1 ) blue dextran (2000 kDa), 2 ) thyroglobulin (669 kDa), 3 ) apoferritin (443 kDa), 4 ) alcohol dehydrogenase (150 kDa), and 5 ) carbonic anhydrase (29 kDa). Fifty milligrams of purified Lf, 100 mg of total milk protein, and 100 mg of total proteins of nonlactating mammary secretions were loaded. Broken vertical lines indicate fraction 52 and represent the elution fraction of the peak of purified Lf in this gel filtration system. OD = Optical density.
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51, and Ig was observed primarily in fractions 43 to 53 (Figure 3b). Similar results were obtained from chromatography of mammary secretions collected on d 14 of involution (Figure 4). In that case, the chromatogram had three major peaks (Figure 4a). The protein profile of fractions of mammary secretions from d 14 of involution indicated that Lf was present in fractions ranging from 35 to 53; MM ranged from 1000 to 80 kDa (Figure 4b). This result was confirmed by immunoblotting developed against anti-bovine Lf (Figure 4c). Chromatography of mammary secretions from d 21 of involution resulted in one major protein peak (Figure 5a). Nevertheless, Lf was observed in fractions 37 to 54; MM ranged from 800 to 80 kDa (Figure 5, b and c). Relatively little casein was present in mammary secretions collected on d 14 or d 21 (Figure 4b and 5b, respectively).
Figure 3. Analysis of proteins in nonlactating mammary secretions from d 7 of involution. a ) Gel filtration chromatogram of protein in nonlactating mammary secretions (pooled sample representing secretions from eight cows; 100 mg of total protein were chromatographed). Vertical lines on the chromatogram indicate fractions that were used for SDS-PAGE analysis. OD = Optical density. b ) SDS-PAGE of proteins in fractions collected from gel filtration chromatography. Lanes are molecular mass standards ( S ) (in kilodaltons; left side of gel), original sample ( O ) , and 22 to 63 (fractions 22 to 63, respectively). Arrows indicate lactoferrin (Lf); serum albumin (SA); Ig, heavy chain (Ig-h); caseins (CN); Ig, light chain (Ig-l); and b-LG. c ) Lactoferrin immunoblot of gel as in panel b. Intact Lf is indicated by the arrow; other bands are proteolytic hydrolysis products of Lf. Journal of Dairy Science Vol. 81, No. 7, 1998
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Of the Lf measured in gel filtration fractions of mammary secretions from d 21 of involution (Figure 6b), approximately 94% was found in fractions 41 to 51; the peak of Lf represented MM approximately 250 kDa. Significant Lf was present in fractions 46 and 49, representing MM of approximately 300 and 150 kDa, respectively. The major peak of purified Lf (80 kDa) was located primarily in fraction 52 on the gel filtration chromatogram (Figure 2b). However, peak fractions containing Lf from both d 7 and 21 of involution were observed in several fractions with higher MM, including fractions 49, 47, 46, and 38, corresponding to MM of approximately 150, 250, 300, and 800 kDa, respectively. The majority of Lf (97 and 99% of total Lf for d 7 and 21, respectively) was present in fractions
Figure 4. Analysis of proteins in nonlactating mammary secretions from d 14 of involution. a ) Gel filtration chromatogram of protein in nonlactating mammary secretions (pooled sample representing secretions from eight cows; 100 mg of total protein were chromatographed). Vertical lines on the chromatogram indicate fractions that were used for SDS-PAGE analysis. OD = Optical density. b ) SDS-PAGE of proteins in fractions collected from gel filtration chromatography. Lanes are molecular mass standards ( S ) (in kilodaltons; left side of gel), original sample ( O ) , and 22 to 64 (fractions 22 to 64, respectively). Arrows indicate lactoferrin (Lf); serum albumin (SA); Ig, heavy chain (Ig-h); caseins (CN); Ig, light chain (Ig-l), and b-LG. c ) Lactoferrin immunoblot of gel as in panel b. Intact Lf is indicated by the arrow; other bands are proteolytic hydrolysis products of Lf.
Quantificaton of Lf in Fractions The Lf content in gel filtration chromatography fractions of mammary secretions from d 7 and 21 of involution was determined by ELISA (Figure 6). Lactoferrin was distributed throughout most of the peaks with higher MM on the gel filtration chromatogram for both d 7 and 21 of involution. In mammary secretions from d 7 of involution (Figure 6a), approximately 75% of total measured Lf was found in fractions 41 to 51; the peak of Lf represented a MM of approximately 250 kDa. Additionally, the shoulder on the descending side of the major Lf peak (fraction 49) contained Lf and had a MM of approximately 150 kDa. About 22% of total Lf was found in fractions 36 to 40; the peak of Lf represented MM of approximately 800 kDa. In total, 97% of total Lf was present in MM fractions that were greater than the 80-kDa monomeric Lf form (fraction 52). Journal of Dairy Science Vol. 81, No. 7, 1998
Figure 5. Analysis of proteins in nonlactating mammary secretions from d 21 of involution. a ) Gel filtration chromatogram of protein in nonlactating mammary secretions (secretions from one of two cows at d 21 of involution that were tested by gel filtration; 100 mg of total protein were chromatographed). Vertical lines on the chromatogram indicate fractions that were used for SDS-PAGE analysis. OD = Optical density. b ) SDS-PAGE of proteins in fractions collected from gel filtration chromatography. Lanes are molecular mass standards ( S ) (in kilodaltons; left side of gel), original sample ( O ) , and 22 to 62 (fractions 22 to 62, respectively). Arrows indicate lactoferrin (Lf); serum albumin (SA); Ig, heavy chain (Ig-h); Ig, light chain (Ig-l); and b-LG. c ) Lactoferrin immunoblot of gel as in panel b. Intact Lf is indicated by the arrow; other bands are proteolytic hydrolysis products of Lf.
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Figure 6. Protein distribution and lactoferrin ( L f ) content in fractions of nonlactating mammary secretions separated by gel filtration chromatography. a ) Chromatogram of mammary secretions from d 7 of involution (pooled sample representing secretions from eight cows; 100 mg of total protein were chromatographed). b ) Chromatogram of mammary secretions from d 21 of involution (secretions from one of two cows at d 21 of involution that were tested by gel filtration; 100 mg of total protein were chromatographed). Solid lines represent optical densities ( O D ) at 280 nm; dashed lines represent Lf contents per fraction as determined by ELISA. The broken vertical line indicates fraction 52 and represents the elution fraction of the peak of purified Lf in this gel filtration system.
representing MM forms greater than monomeric Lf. In addition, 75% of Lf on d 7 and 94% of Lf on d 21 were present in fractions that also contained Ig (Figures 3b and 5b); 22% of total Lf on d 7 was present in fractions containing casein. These percentages were based on the protein profiles by SDSPAGE. DISCUSSION Results from both gel filtration chromatography and sucrose density gradient ultracentrifugation indicated that Lf exists in protein complexes with high MM in nonlactating mammary secretions during involution. These Lf complexes have MM generally greater than 80 kDa and up to 1100 kDa. Coseparation of Lf with other major proteins suggests that Lf may complex with casein, Ig, or both in nonlactating mammary secretions, resulting in the apparent shift of the MM of Lf. The relative absence of casein in mammary secretions from d 14 and 21 of involution suggests that Lf may be complexing primarily with Ig or with some other component to result in separation
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in fractions with higher MM. Quantification of Lf in fractions from gel filtration chromatography indicated that the majority of Lf was present in complexes with higher MM rather than in the monomeric form. During early involution, 97% of total Lf is present in forms with high MM. Seventy-five percent of total Lf is present in fractions that contain Ig, and much of the remainder is present in fractions that contain casein. As involution progresses, there is an increase in the proportion of Lf present in fractions with MM of approximately 250 kDa. The presence of Lf with high MM complexes in the nonlactating mammary secretions is consistent with the findings of other investigators. Isolated human Lf has a pronounced tendency to become associated with acidic proteins, such as albumin or casein, and these interactions influence its electrophoretic behavior in agarose gels (10). Lactoferrin in bovine milk has also been found to be associated with other milk proteins ( 8 ) . A gel filtration fraction with a high MM (less than 500 kDa) from nonlactating mammary secretions contained Lf and casein (20). Results from the present study indicated that most of the Lf that is present in mammary secretions during d 7 and 21 of involution separates in fractions with a MM of approximately 250 kDa. The 250-kDa fractions containing Lf also contain Ig. The presence of Lf in fractions representing 150 kDa might suggest the presence of Lf dimers, and the 300-kDa fractions might suggest the presence of Lf tetramers or aggregates of two Lf molecules plus one Ig molecule. These results are also consistent with the observation that the apparent mean MM of Lf, determined by gel filtration, increases during mastitis ( 9 ) . In that case, Lf separated at approximately the size of a trimer as the infection progressed, concurrent with increasing Lf concentrations. The nature of the binding interactions that result in the formation of Lf complexes in nonlactating mammary secretions is unknown. Ionic, disulfide, and hydrophobic interactions may all be involved in the binding of Lf with other macromolecules. The binding of Lf to a monomeric IgA affinity column can be dissociated with increasing salt concentrations (22), which suggests that the binding between Lf and IgA is an ionic interaction. Lactoferrin is a strong basic protein at a neutral pH. Casein mainly exists as casein micelles that contain phosphate groups. A possible explanation of the interaction between Lf and casein may be via an ionic interaction. In addition to ionic interactions, other forces also appear to be involved in the formation of complexes. For example, disulfide reduction will separate both the secretory Journal of Dairy Science Vol. 81, No. 7, 1998
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component and Lf from IgA preparations, suggesting that disulfide bond formation may be involved in this binding interaction (28). Lysozyme is also a strong basic protein and is positively charged at a neutral pH. Hydrophobic interactions are thought to be responsible for the formation of a Lf-lysozyme complex because it is not dissociated by ion-exchange chromatography ( 5 ) . The binding relationship between Lf and Ig may involve ionic, hydrophobic, disulfide, or multiple interactions in mammary secretions. Furthermore, human Lf can undergo polymerization in fluids containing calcium, resulting in tetramer formation, which is the predominant molecular form in human serum, tears, and breast milk ( 2 ) . A complex of Lf-IgG2 was identified in colostrum by Butler ( 4 ) and was estimated at greater than 700 kDa by gel filtration chromatography. Results from the present study indicate that the MM of Lf complexes in nonlactating mammary secretions range up to 1100 kDa. This range of Lf complexes with high MM may result from the different molar ratios of Lf to Ig or Lf to casein. The size of the Lf-IgG2 complex suggests that multiple molecules of both proteins may form the aggregate ( 4 ) . A complex of Lf and lysozyme of about 110 kDa has been observed in human milk (13). However, there is very little lysozyme in bovine mammary secretions, and such a Lf-lysozyme complex is unlikely to contribute significantly to the findings of the present study. Groves ( 7 ) found significant amounts of Lf in both the acid-precipitated casein fraction and in the whey fraction from normal milk. However, little subsequent work has been done to quantify the distribution of free Lf and complexed Lf in the mammary secretions. This scenario led to the second objective of the present study, which was to determine the proportion of free Lf and complexed Lf in mammary secretions during involution. The major proportion of total Lf exists in complexes with high MM rather than as monomeric Lf. A majority of Lf exists in fractions representing a range in MM from 150 to 300 kDa. However, in secretions from d 7 of involution, approximately 22% of total Lf is also present in a peak with the MM around 800 kDa and cochromatographed with casein. As the proteolytic activity increases during involution, casein is progressively degraded ( 1 ) . Therefore, the proportion of Lf-casein complexes would be expected to decrease by d 21 of involution compared with d 7 of involution. Lactoferrin-Ig or LfLf multimers probably are the major Lf complex forms in nonlactating mammary secretions by d 21 of involution. This large proportion of Lf complexes suggests that Lf-Ig complexes or Lf-Lf multimers may be important during involution and important in the Journal of Dairy Science Vol. 81, No. 7, 1998
resistance of the bovine mammary gland to IMI during that period. The biological relevance of Lf complexes to the functions of Lf, Ig, casein, or lysozyme remains unclear. The bacteriostatic activity of milk has been attributed primarily to the presence of Lf (18, 21, 24). Complexes of Lf with other proteins or with itself might either enhance or reduce the antibacterial activity of Lf. Little evidence is available to show that Lf complexes, such as Lf-Ig or Lf-Lf multimers, enhance the antibacterial activity in mammary secretions. However, human Lf and IgA act cooperatively with increased bacteriostatic activity (26). Immunoglobulin A alone shows some inhibition of bacterial growth, but the inhibition is considerably increased in the presence of Lf. In contrast, Lf may bind to casein micelles and form Lf-casein complexes. This interaction might prevent the interaction between Lf and lipopolysaccharide on the surface of bacteria, which is one of the Lf antibacterial mechanisms (6, 12). As a consequence, the complexing of Lf with casein might decrease the antibacterial activity of Lf. Alternatively, fractions with high MM from nonlactating mammary secretions that contained both Lf and casein were found to inhibit growth of Bacillus species (20). The nature of the Lf complexes and the intermolecular interactions of Lf with other proteins found in mammary secretions requires further characterization. CONCLUSIONS Results from gel filtration chromatography and sucrose density gradient ultracentrifugation indicate that Lf is present in complexed forms with high MM in nonlactating mammary secretions. A majority of total Lf in nonlactating mammary secretions exists in fractions with a MM of approximately 250 kDa. A smaller proportion of total Lf exists in fractions containing casein, representing a MM of approximately 800 kDa. The apparent shift in the MM of Lf from the monomeric form may be due to the presence of Lf complexes such as Lf-casein, Lf-Ig, or Lf-Lf multimers in mammary secretions. As involution progresses, the proportion of Lf as Lf-Ig complexes, and Lf-Lf multimers apparently increase. Identification of Lf in complexed forms in mammary secretions may lead to a reevaluation of the relative importance of Lf as a host defense factor in the mammary gland. ACKNOWLEDGMENTS The authors acknowledge the assistance of M. Aslam in sample collection and processing. We also appreciate the technical expertise and assistance of H. Hegarty and S. Zou in this work.
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Journal of Dairy Science Vol. 81, No. 7, 1998