Studies of the structure of the metastasis-associated 67 kDa laminin binding protein: Fatty acid acylation and evidence supporting dimerization of the 32 kDa gene product to form the mature protein

Terry H Landowski, E. A. Dratz, J. R. Starkey

Research output: Contribution to journalArticle

96 Citations (Scopus)

Abstract

The level of expression of the 67 kDa high-affinity laminin binding protein (LBP) correlates with the progression of many solid tumors. The cDNA clone for the 67 kDa LBP is sufficient to encode a polypeptide of only 32 kDa, and there is no readily identifiable mechanism for membrane association. We have overexpressed the transfected 67 kDa hamster LBP in quantities that have enabled us to analyze the membrane-bound form of the protein. Treatment of the purified LBP with methyl transesterification reagents, followed by GC- MS, identified the covalently bound fatty acids palmitate, stearate, and oleate. The fatty acid modification may provide a mechanism for membrane association. Molecular mass determination by MALDI-TOF MS demonstrated the true molecular mass of the protein to be 66.7 kDa, compatible with the SDS- PAGE observation of 67 kDa. Treatment of the LBP with neuraminidase, O- glycanase, or Endo-F glycosidase has no detectable effect on the apparent molecular mass of the protein, and the MALDI-TOF MS did not show evidence of mass heterogeneities typically observed with glycosylated proteins. Reduction with dithiothreitol or β-mercaptoethanol had no effect on the apparent molecular mass on SDS-PAGE or on the relative quantities of molecular mass species on MALDI-TOF MS. The experimentally determined amino acid composition, however, was found to be consistent with the 67 kDa form being a homodimer of the 32 kDa precursor. Preliminary experiments also suggest that the high-affinity laminin binding characteristic of the protein may be modulated by an, as yet, unidentified membrane accessory molecule.

Original languageEnglish (US)
Pages (from-to)11276-11287
Number of pages12
JournalBiochemistry
Volume34
Issue number35
DOIs
StatePublished - 1995
Externally publishedYes

Fingerprint

Acylation
Fatty Acid-Binding Proteins
Dimerization
Laminin
Molecular mass
Carrier Proteins
Genes
Neoplasm Metastasis
Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry
Membranes
Proteins
Polyacrylamide Gel Electrophoresis
Fatty Acids
Association reactions
Stearates
Mercaptoethanol
Palmitates
Dithiothreitol
Glycoside Hydrolases
Transesterification

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{d23a6da13660413b8c5ecc8f95ee4cab,
title = "Studies of the structure of the metastasis-associated 67 kDa laminin binding protein: Fatty acid acylation and evidence supporting dimerization of the 32 kDa gene product to form the mature protein",
abstract = "The level of expression of the 67 kDa high-affinity laminin binding protein (LBP) correlates with the progression of many solid tumors. The cDNA clone for the 67 kDa LBP is sufficient to encode a polypeptide of only 32 kDa, and there is no readily identifiable mechanism for membrane association. We have overexpressed the transfected 67 kDa hamster LBP in quantities that have enabled us to analyze the membrane-bound form of the protein. Treatment of the purified LBP with methyl transesterification reagents, followed by GC- MS, identified the covalently bound fatty acids palmitate, stearate, and oleate. The fatty acid modification may provide a mechanism for membrane association. Molecular mass determination by MALDI-TOF MS demonstrated the true molecular mass of the protein to be 66.7 kDa, compatible with the SDS- PAGE observation of 67 kDa. Treatment of the LBP with neuraminidase, O- glycanase, or Endo-F glycosidase has no detectable effect on the apparent molecular mass of the protein, and the MALDI-TOF MS did not show evidence of mass heterogeneities typically observed with glycosylated proteins. Reduction with dithiothreitol or β-mercaptoethanol had no effect on the apparent molecular mass on SDS-PAGE or on the relative quantities of molecular mass species on MALDI-TOF MS. The experimentally determined amino acid composition, however, was found to be consistent with the 67 kDa form being a homodimer of the 32 kDa precursor. Preliminary experiments also suggest that the high-affinity laminin binding characteristic of the protein may be modulated by an, as yet, unidentified membrane accessory molecule.",
author = "Landowski, {Terry H} and Dratz, {E. A.} and Starkey, {J. R.}",
year = "1995",
doi = "10.1021/bi00035a037",
language = "English (US)",
volume = "34",
pages = "11276--11287",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "35",

}

TY - JOUR

T1 - Studies of the structure of the metastasis-associated 67 kDa laminin binding protein

T2 - Fatty acid acylation and evidence supporting dimerization of the 32 kDa gene product to form the mature protein

AU - Landowski, Terry H

AU - Dratz, E. A.

AU - Starkey, J. R.

PY - 1995

Y1 - 1995

N2 - The level of expression of the 67 kDa high-affinity laminin binding protein (LBP) correlates with the progression of many solid tumors. The cDNA clone for the 67 kDa LBP is sufficient to encode a polypeptide of only 32 kDa, and there is no readily identifiable mechanism for membrane association. We have overexpressed the transfected 67 kDa hamster LBP in quantities that have enabled us to analyze the membrane-bound form of the protein. Treatment of the purified LBP with methyl transesterification reagents, followed by GC- MS, identified the covalently bound fatty acids palmitate, stearate, and oleate. The fatty acid modification may provide a mechanism for membrane association. Molecular mass determination by MALDI-TOF MS demonstrated the true molecular mass of the protein to be 66.7 kDa, compatible with the SDS- PAGE observation of 67 kDa. Treatment of the LBP with neuraminidase, O- glycanase, or Endo-F glycosidase has no detectable effect on the apparent molecular mass of the protein, and the MALDI-TOF MS did not show evidence of mass heterogeneities typically observed with glycosylated proteins. Reduction with dithiothreitol or β-mercaptoethanol had no effect on the apparent molecular mass on SDS-PAGE or on the relative quantities of molecular mass species on MALDI-TOF MS. The experimentally determined amino acid composition, however, was found to be consistent with the 67 kDa form being a homodimer of the 32 kDa precursor. Preliminary experiments also suggest that the high-affinity laminin binding characteristic of the protein may be modulated by an, as yet, unidentified membrane accessory molecule.

AB - The level of expression of the 67 kDa high-affinity laminin binding protein (LBP) correlates with the progression of many solid tumors. The cDNA clone for the 67 kDa LBP is sufficient to encode a polypeptide of only 32 kDa, and there is no readily identifiable mechanism for membrane association. We have overexpressed the transfected 67 kDa hamster LBP in quantities that have enabled us to analyze the membrane-bound form of the protein. Treatment of the purified LBP with methyl transesterification reagents, followed by GC- MS, identified the covalently bound fatty acids palmitate, stearate, and oleate. The fatty acid modification may provide a mechanism for membrane association. Molecular mass determination by MALDI-TOF MS demonstrated the true molecular mass of the protein to be 66.7 kDa, compatible with the SDS- PAGE observation of 67 kDa. Treatment of the LBP with neuraminidase, O- glycanase, or Endo-F glycosidase has no detectable effect on the apparent molecular mass of the protein, and the MALDI-TOF MS did not show evidence of mass heterogeneities typically observed with glycosylated proteins. Reduction with dithiothreitol or β-mercaptoethanol had no effect on the apparent molecular mass on SDS-PAGE or on the relative quantities of molecular mass species on MALDI-TOF MS. The experimentally determined amino acid composition, however, was found to be consistent with the 67 kDa form being a homodimer of the 32 kDa precursor. Preliminary experiments also suggest that the high-affinity laminin binding characteristic of the protein may be modulated by an, as yet, unidentified membrane accessory molecule.

UR - http://www.scopus.com/inward/record.url?scp=0029154305&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0029154305&partnerID=8YFLogxK

U2 - 10.1021/bi00035a037

DO - 10.1021/bi00035a037

M3 - Article

C2 - 7669786

AN - SCOPUS:0029154305

VL - 34

SP - 11276

EP - 11287

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 35

ER -