Haematologic Technologies, Inc. - Lactadherin

THE STRUCTURE OF LACTADHERIN Lactadherin binds to phosphatidyl-L-serine via the C2 domain and binds the avb3 and avb5 integrins via the second EGF domain. The one letter amino acid codes refer to identified amino acids that participate in binding. For the C2 domain the capital letters refer to water-exposed hydrophobic residues.

PURCHASING AND PRODUCT INFORMATION

Catalog Number

BLAC-1200

BLAC-FITC

Description

Bovine Lactadherin

FITC-labeled B.Lact.

Size

50 µg

1 mL (1.6 micromolar)

Formulation

70 mM Sodium Phosphate, pH 7.0

TBS, 1% BSA, 0.02% azide, pH 7.4

Storage

-70^oC (BLAC-1200)

4^oC (BLAC-FITC)

Purity

>95% by SDS-PAGE (both)

Activity Determination

N/A

Shelf Life (properly stored)

1 year (both)

Sample Gel Information:

Gel: Novex 4-12% Bis-Tris

Load: Bovine Lactadherin, 1 µg per lane

Buffer: MOPS

Standard: SeeBluePlus 2; Myosin (191 kDa), Phosphorylase B (97 kDa), BSA (64 kDa), Glutamic Dehydrogenase (51 kDa), Alcohol Dehydrogenase (39 kDa), Carbonic Anhydrase (28 kDa), Myoglobin Red (19 kDa), Lysozyme (14 kDa)

U.S. Pricing

Product inquiry

ORDER NOW!

Overview of Lactadherin

Lactadherin is a widely distributed glycoprotein (~ 50 kDa), which was originally characterized due to its association with milk fat/lipid globule membranes. Synonymous names are PAS-6/7, bovine-associated mucoprotein, BA-46, P47, and MFG-E8. Structural hallmarks of lactadherin are the presence of two epidermal growth factor (EGF) homology domains (with an RGD peptide motif in the second EGF domain), and two C domains sharing homology with the discoidin family of lectin domains including the phospholipid-binding domains of blood clotting factors V and VIII. Lactadherin shows preferential binding to phosphatidylserine (L-form) in a calcium independent manner.

Purified lactadherin functions as an anticoagulant by blocking phosphtidylserine-containing membrane sites for blood coagulation proteins (10). Fluoresence-labeled lactadherin functions as a sensitive probe for exposed phosphtidylserine on nucleated cells and on stimulated platelets (8, 9) . Lactadherin will bind to membranes that have phosphatatidylserine content below the threshold for annexin V binding.

Lactadherin is purified from un-pasteurized bovine milk (11).

Illustrated Applications

Above: K562 cells (left) and HL60 cells (right) co-stained with both FITC-conjugated lactadherin (green) and Alexa-647 conjugated annexin V (red) early in apoptosis. The annexin is internalized in granules and is not detectably staining the cells. Reference: Shi, J., Y. Shi, L. N. Waehrens, J. T. Rasmussen, C. W. Heegaard and G. E. Gilbert (2006). "Lactadherin detects early phosphatidylserine exposure on immortalized leukemia cells undergoing programmed cell death." Cytometry A 69(12): 1193-201. Copyright 2006. John Wiley & Sons, Inc. Reprinted with permission of John Wiley & Sons, Inc.

Above: HeLa cells stained with FITC-conjugated lactadherin 2 hours (left) and 3 hours (right) after treatment with staurosporine. Early in apoptosis the cells have small vesicles and long, thin appendages that stain avidly for lactadherin. Reference: Waehrens LN, Heeghaard, CW, Gilbert GE, Rasmussen JT. Bovine Lactadherin as a Calcium-independent Imaging Agent of Phosphatidylserine Expressed on the Surface of Apoptotic HeLa Cells 2009 J. Histochem. Cytochem. (ePub June 2009).

Above: Phosphtidylserine exposure in mouse mesenteric venous thrombosis. Mice were given 1 µg each of lactadherin and annexin V by tail vein immediately prior to externalization of the mesentery. The mesentary was exposed to ferric chloride and then the animals were perfused with saline/paraformaldehyde. Serial sections of the mesentary were stained with anti-fibrinogen/fibrin (left), anti-platelet (middle), and anti-lactadherin antibodies (right) developed with the alkaline phosphatase Vector Red substrate. A layer of fibrinogen/fibrin (left, closed arrows) overlaid a mural hemorrhage (open star). Platelets (middle) were scattered along the luminal surface of the thrombus (open triangles) as well as upon fibrinogen/fibrin strands extending into the lumen. Lactadherin staining (right) was strongest along the raised endothelium surface (closed arrow), including adherent platelets close to the wall. Platelets on fibrin strands did not stain detectably (open arrow). (Shi J, Pipe SW, Rasmussen JT, Heegaard CW, Gilbert GE. Lactadherin blocks thrombosis and hemostasis in vivo: correlation with platelet phosphatidylserine exposure. J Thromb Haemost. Jul 2008;6(7):1167-1174).

Properties of Lactadherin

Names:

Lactadherin, MFG-E8, PAS-6/7, SED1, p47

Localization:

Milk (1) , also secreted by stimulated macrophages (2) , vascular smooth muscle (3) , endothelial cells (4)

Plasma concentration:

unknown in normal adults, measurable in women with metastatic breast cancer (5) , measurable in newborn calves after milk meal (1)

Mode of action:

Physiologic function as opsonin or bridge ligand (6, 7) . The lectin-like domains engage phosphatidyl-L-serine, the second EGF domain engages avb3 and/or avb5 integrin leading to phagocytosis of apoptotic cells.

Molecular weight:

52 kDa and 47 kDa (glycosylation variants, PAS-6 and PAS-7 respectively)

Extinction coefficient:

1 %

1 c m, 280 nm

= 16.5 (calculated)

Structure:

single chain with two EGF domains and two C domains. The mouse isoform contains a central proline rich region and the human molecule contains a single EGF domain

Percent carbohydrate:

6%, 13% for glycosylation variants

Post-translational modification:

glycosylation

References

Butler, J.E., Pringnitz, D.J., Martens, C.L., and Crouch, N. 1980. Bovine-associated mucoprotein: I. Distribution among adult and fetal bovine tissues and body fluids. Differentiation 17:31-40.
Hanayama, R., Tanaka, M., Miwa, K., Shinohara, A., Iwamatsu, A., and Nagata, S. 2002. Identification of a factor that links apoptotic cells to phagocytes. Nature 417:182-187.
Haggqvist, B., Naslund, J., Sletten, K., Westermark, G.T., Mucchiano, G., Tjernberg, L.O., Nordstedt, C., Engstrom, U., and Westermark, P. 1999. Medin: an integral fragment of aortic smooth muscle cell-produced lactadherin forms the most common human amyloid. Proc Natl Acad Sci U S A 96:8669-8674.
Silvestre, J.S., Thery, C., Hamard, G., Boddaert, J., Aguilar, B., Delcayre, A., Houbron, C., Tamarat, R., Blanc-Brude, O., Heeneman, S., et al. 2005. Lactadherin promotes VEGF-dependent neovascularization. Nat Med 11:499-506.
Ceriani, R.L., Sasaki, M., Sussman, H., Wara, W.M., and Blank, E.W. 1982. Circulating human mammary epithelial antigens in breast cancer. Proc Natl Acad Sci U S A 79:5420-5424.
Hanayama, R., Tanaka, M., Miyasaka, K., Aozasa, K., Koike, M., Uchiyama, Y., and Nagata, S. 2004. Autoimmune disease and impaired uptake of apoptotic cells in MFG-E8-deficient mice. Science 304:1147-1150.
Hanayama, R., and Nagata, S. 2005. Impaired involution of mammary glands in the absence of milk fat globule EGF factor 8. Proc Natl Acad Sci U S A 102:16886-16891.
Shi, J., Shi, Y., Waehrens, L.N., Rasmussen, J.T., Heegaard, C.W., and Gilbert, G.E. 2006. Lactadherin detects early phosphatidylserine exposure on immortalized leukemia cells undergoing programmed cell death. Cytometry A 69:1193-1201.
Dasgupta, S.K., Guchhait, P., and Thiagarajan, P. 2006. Lactadherin binding and phosphatidylserine expression on cell surface-comparison with annexin A5. Transl Res 148:19-25.
Shi, J., and Gilbert, G.E. 2003. Lactadherin inhibits enzyme complexes of blood coagulation by competing for phospholipid binding sites. Blood 101:2628-2636.
Hvarregaard et al., 1996. Eur. J. Biochem. 240.

Please inquire about products not listed. We can also custom purify proteins from other species.