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Carbohydrate-binding profile of a pregnancy-associated rat uterine glycoprotein

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Abstract

Sugar-binding proteins obtained from the peri-implantation uterine tissue have been thought in recent years to have significant roles in embryo implantation, where carbohydrate moieties of the protein are actively involved. Based on this rationale a mannose-containing glycoprotein/lectin (named uterine agglutinin or UA) was purified by Concanavalin A (Con A) affinity chromatography in a previous study. A modification of the original purification procedure to include a 33% ammonium sulfate fractionation improves the yield of the protein significantly. An alternative purification procedure by Mannan affinity matrix, indicates that apart from containing mannose, UA possesses mannose-binding properties as well.

In this paper, we report some of the biochemical and more specifically, the carbohydrate-binding characteristics of UA. The protein is seen to contain mannose-6-phosphate (M-6-P)-binding sites, which is of importance since M-6-P receptors have a large number of biologically significant roles, including that of binding to growth factors.

SDS-PAGE, gel filtration chromatography and alkaline PAGE indicate the homogenous nature of the protein with subunit molecular weights of 36 kDa and 19 kDa, and a native size of 64kDa. Amino acid analysis shows glycine, glutamic acid and aspartic acid to be the major constituents.

UA is a glycoprotein and shows presence of N-acetyl glucosamine and galactose, apart from mannose.De nove synthesis studies in the presence of tunicamycin show that the carbohydrate moiety of the glycoprotein is attached by N-linkage to the protein. Binding characteristics of the protein is studied quantitatively in which (125I)-labelled lectin is bound to Mannan-Sepharose affinity matrix. The sugar inhibition pattern of this binding shows α-methyl mannopyranoside and M-6-P to be equally effective as inhibitors. Scatchard analysis of the binding of UA to (14C)-mannose shows a Ka of 6.43×105 (M−1) and that 1 mole of UA can bind to 8 moles of mannose. The possible role of the protein in implantation has also been discussed.

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Abbreviations

b.w.:

body weight

BSA:

Bovine Serum Albumin

Con A:

Concanavalin A

cpm:

counts per minute

Endo H:

endoglycosidase H

GlcNAc:

N-acetyl glucosamine

Man:

mannose

M-6-P:

mannose-6-phosphate

MEM-deficient:

Minimum Essential Medium Eagle-deficient modification

NaBH4 :

sodium borohydride

NaN3 :

sodium azide

(NH4)2SO4 :

ammonium sulphate

p.c.:

post coitum

PMSF:

phenyl methyl sulphonyl fluoride

PTA:

phosphotungstic acid

RCA:

Ricinus communis Agglutinin

SDS-PAGE:

sodium dodecyl sulphate-polyacrylamide gel electrophoresis

TCA:

trichloroacetic acid

UA:

Uterine Agglutinin

WGA:

Wheat-germ Agglutinin

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Authors and Affiliations

  1. Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Jadavpur, 700 032, Calcutta, India

    Monika Das, Prabir K. Mukhopadhyay & Mridula Chowdhury

Authors
  1. Monika Das
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  2. Prabir K. Mukhopadhyay
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  3. Mridula Chowdhury
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Das, M., Mukhopadhyay, P.K. & Chowdhury, M. Carbohydrate-binding profile of a pregnancy-associated rat uterine glycoprotein. Mol Cell Biochem 137, 91–99 (1994). https://doi.org/10.1007/BF00944070

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